OBG3 Globular Head and Uses Thereof

ABSTRACT

The present invention relates to the field of obesity research. Obesity is a public health problem that is serious and widespread. A compound, globular OBG3, has been identified that reduces weight gain in animals. This compound should be effective for reducing body mass and for treating obesity-related diseases and disorders. These obesity-related diseases and disorders include hyperlipidermias, atherosclerosis, diabetes, and hypertension.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 11/132,814,filed May 19, 2005, which is a continuation-in-part of U.S. applicationSer. No. 10/285,833, filed Nov. 1, 2002, now U.S. Pat. No. 6,989,367,which is a continuation-in-part of U.S. application Ser. No. 09/909,547,filed Jul. 19, 2001, now U.S. Pat. No. 6,579,852, which is acontinuation-in-part of U.S. application Ser. No. 09/776,976, filed Feb.5, 2001, now U.S. Pat. No. 6,566,332, which is a continuation-in-part ofU.S. application Ser. No. 09/758,055, filed Jan. 10, 2001, abandoned,all of which claim the benefit of U.S. Provisional application Ser. No.60/176,228, filed Jan. 14, 2000, U.S. Provisional application Ser. No.60/198,087, filed Apr. 13, 2000, and U.S. Provisional application Ser.No. 60/229,881, filed Sep. 1, 2000. This application is also acontinuation-in-part of U.S. application Ser. No. 10/231,814, filed Aug.29, 2002, now U.S. Pat. No. 6,967,091, which is a continuation of U.S.application Ser. No. 09/758,055, filed Jan. 10, 2001, abandoned, both ofwhich claim the benefit of U.S. Provisional application Ser. No.60/176,228, filed Jan. 14, 2000, U.S. Provisional application Ser. No.60/198,087, filed Apr. 13, 2000, and U.S. Provisional application Ser.No. 60/229,881, filed Sep. 1, 2000. U.S. application Ser. No. 10/285,833claims the benefit of U.S. Provisional application Ser. No. 60/340,077,filed Nov. 1, 2001, U.S. Provisional application Ser. No. 60/355,478,filed Feb. 6, 2002, and U.S. Provisional application Ser. No.60/355,563, filed Feb. 6, 2002.

FIELD OF THE INVENTION

The present invention relates to the field of metabolic research, inparticular the discovery of compounds effective for reducing body massand useful for treating obesity-related diseases and disorders. Theobesity-related diseases or disorders envisioned to be treated by themethods of the invention include, but are not limited to,hyperlipidemia, atherosclerosis, diabetes, and hypertension.

BACKGROUND OF THE INVENTION

The following discussion is intended to facilitate the understanding ofthe invention, but is not intended nor admitted to be prior art to theinvention.

Obesity is a public health problem that is serious, widespread, andincreasing. In the United States, 20 percent of the population is obese;in Europe, a slightly lower percentage is obese [Friedman (2000) Nature404:632-634]. Obesity if associated with increased risk of hypertension,cardiovascular disease, diabetes, and cancer as well as respiratorycomplications and osteroarthritis [Kopelman (2000) Nature 404:635-643].Even modest weight loss ameliorates these associated conditions.

While still acknowledging that lifestyle factors including environment,diet, age and exercise play a role in obesity, twin studies, analyses offamilia aggregation, and adoption studies all indicate that obesity islargely the result of genetic factors [Barsh et al. (2000) Nature 404:644-651]. In agreement with these studies, is the fact that anincreasing number of obesity-related genes are being identified. Some ofthe more extensively studied genes include those encoding leptin (ob)and its receptor (db), pro-opiomelanocortin (Pomc),melanocortin-4-receptor (Mc4r), agouti protein (A^(y)), carboxypeptidaseE (fat), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basichelix-loop-helix 2 (Nhlh2), prohormone convertase 1 (PCSK1), and tubbyprotein (tubby) [rev'd in Barsh et al. (2000) Nature 404: 644-651].

SUMMARY OF THE INVENTION

The instant invention is based on the discovery that portions of thefull-length OBG3 polypeptide, termed OBG3 polypeptide fragments or gOBG3polypeptide fragments, have unexpected effects in vitro and in vivo,including utility for weight reduction, prevention of weight gain, andcontrol of blood glucose levels in humans and other mammals. Theseunexpected effects of OBG3 or gOBG3 polypeptide fragment administrationin mammals also include reduction of elevated free fatty acid levelscaused by administration of epinephrine, i.v. injection of “intralipid”,or administration of a high fat test meal, as well as increased fattyacid oxidation in muscle cells, and weight reduction in mammalsconsuming a high fat/high sucrose diet. These effects are unexpected andsurprising given that administration of full-length OBG3 polypeptidetypically has no effect or a significantly reduced effect in vivo or invitro depending on the specific biological activity and the amountadministered. To the extent that any effect is observed followingadministration of full-length OBG3 polypeptide, the levels offull-length OBG3 polypeptide required for an effect render it unfeasiblein most instances as a potential treatment for humans at this time. Incontrast, the OBG3 and gOBG3 polypeptide fragments of the invention areradically more effective and thus can be provided at levels that arefeasible for treatments in humans.

Thus, the invention is drawn to OBG3 and gOBG3 polypeptide fragments,polynucleotides encoding said OBG3 and gOBG3 polypeptide fragments,vectors comprising said OBG3 and gOBG3 polynucleotides, and cellsrecombinant for said OBG3 and gOBG3 polynucleotides, as well as topharmaceutical and physiologically acceptable compositions comprisingsaid OBG3 and gOBG3 polypeptide fragments and methods of administeringsaid OBG3 and gOBG3 pharmaceutical and physiologically acceptablecompositions in order to reduce body weight or to treat obesity-relateddiseases and disorders. Assays for identifying agonists and antagonistsof obesity-related activity are also part of the invention.

In a first aspect, the invention features a purified, isolated, orrecombinant OBG3 or gOBG3 polypeptide fragment that has significantlygreater activity than a full-length OBG3 polypeptide, wherein saidactivity is selected from the group consisting of lipid partitioning,lipid metabolism, and insulin-like activity. In preferred embodiments,said polypeptide fragment comprises, consists essentially of, orconsists of, at least 6 and not more than 238 consecutive amino acids ofSEQ ID NO:6 or at least 6 and not more than 241 consecutive amino acidsof SEQ ID NO:2 or SEQ ID NO:4. In other preferred embodiments, OBG3 orgOBG3 polypeptide fragments having unexpected activity are selected fromamino acids 84-244, 85-244, 86-244, 87-244, 88-244, 89-244, 90-244,91-244, 92-244, 93-244, 94-244, 95-244, 96-244, 97-244, 98-244, 99-244,100-244, 101-244, 102-244, or 103-244 of SEQ ID NO:6. In other preferredembodiments, OBG3 or gOBG3 polypeptide fragments having unexpectedactivity are selected from amino acids 88-247, 89-247, 90-247, 91-247,92-247, 93-247, 94-247, 95-247, 96-247, 97-247, 98-247, 99-247, 100-247,101-247, 102-247, 103-247, 104-247, 105-247, or 106-247 of SEQ ID NO:2or SEQ ID NO:4. In other preferred embodiments, OBG3 or gOBG3polypeptide fragments are selected from amino acids about 84 to 244, 85to 244, 101 to 244, 102 to 244, or 103 to 244 of SEQ ID NO:6 and aminoacids 88 to 247, 104 to 247, 105 to 247, or 106 to 247 of SEQ ID NO:2 orSEQ ID NO:4. In further preferred embodiments, gOBG3 polypeptidefragments are said selected gOBG3 polypeptide fragments made resistantto dipeptidyl peptidase cleavage by N-terminal modification. In otherfurther preferred embodiments, said polypeptide fragment comprises anamino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the correspondingconsecutive amino acids of SEQ ID NO:6, SEQ ID NO:2 or SEQ ID NO:4.

In other highly preferred embodiments, said polypeptide fragmentcomprises, consists essentially of, or consists of, a purified,isolated, or recombinant gOBG3 fragment. Preferably, said gOBG3polypeptide fragment comprises, consists essentially of, or consists of,at least 6 consecutive amino acids of amino acids 84 to 244 of SEQ IDNO:6 or at least 6 consecutive amino acids of amino acids 88 to 247 ofSEQ ID NO:2 or SEQ ID NO:4. In other preferred embodiments, gOBG3polypeptide fragments having unexpected activity are selected from aminoacids 84-244, 85-244, 86-244, 87-244, 88-244, 89-244, 90-244.91-244,92-244, 93-244, 94-244, 95-244, 96-244, 97-244, 98-244, 99-244, 100-244,101-244, 102-244, or 103-244 of SEQ ID NO:6. In other preferredembodiments, gOBG3 polypeptide fragments having unexpected activity areselected from amino acids 88-247, 89-247, 90-247, 91-247, 92-247.93-247,94-247, 95-247, 96-247, 97-247.98-247, 99-247, 100-247, 101-247,102-247, 103-247, 104-247, 105-247, or 106-247 of SEQ ID NO:2 or SEQ IDNO:4. In other preferred embodiments, gOBG3 polypeptide fragments areselected from amino acids 84 to 244, 85 to 244, 101 to 241, 102 to 244,or 103 to 2-4 of SEQ ID NO:6 and amino acids 88 to 247, 104 to 247, 105to 247, or 106 to 247 of SEQ ID NO:2 or SEQ ID NO:4. In furtherpreferred embodiments, gOBG3 polypeptide fragments are said selectedgOBG3 polypeptide fragments made resistant to dipeptidyl peptidasecleavage by N-terminal modification. Alternatively, said gOBG3 fragmentcomprises, consists essentially of, or consists of, an amino acidsequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the corresponding amino acids 84 to 244 of SEQ ID NO:6 orat least 75% identical to amino acids 88 to 247 of SEQ ID NO:2 or SEQ IDNO:4. In a further preferred embodiment, the OBG3 polypeptide fragmentis identical to an APM1 proteolytic cleavage product from human plasma.Preferably, the proteolytic cleavage product comprises the Clq globularhead or a portion thereof. More preferably, the proteolytic cleavageproduct is identical to a proteolytic cleavage product isolated fromhuman plasma by immunoprecipitation using antibodies specific for theClq globular head. More preferably the proteolytic cleavage productcannot be immunoprecipitated from human plasma using an antibodydirected against the human non-homologous region (HDQETTTQGPGVLLPLPKGA)of APM1. Still more preferably, the APM1 proteolytic cleavage producthas an apparent molecular weight of 27 kDa using SDS-PAGE.

In a further preferred embodiment, the OBG3 or gOBG3 polypeptidefragment is able to lower circulating (either blood, serum or plasma)levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or(iii) triglycerides. Further preferred polypeptide fragmentsdemonstrating free fatty acid level lowering activity, glucose levellowering activity, and/or triglyceride level lowering activity, have anactivity that is significantly greater than full-length OBG3 at the samemolar concentration, have a greater than transient activity and/or havea sustained activity.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatsignificantly stimulate muscle lipid or free fatty acid oxidation ascompared to full-length OBG3 polypeptides at the same molarconcentration. Further preferred OBG3 or gOBG3 polypeptide fragments arethose that cause C2C12 cells differentiated in the presence of saidfragments to undergo at least 10%, 20%, 30%, 35, or 40% more oleateoxidation as compared to untreated cells or cells treated withfull-length OBG3.

Further preferred OBG3 or gOBG3 polypeptide fragments are those that areat least 30% more efficient than full-length OBG3 at increasing leptinuptake in a liver cell line (preferably BPRCL mouse liver cells (ATCCCRL-2217)).

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatsignificantly reduce the postprandial increase in plasma free fattyacids, particularly following a high fat meal.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatsignificantly reduce or eliminate ketone body production, particularlyfollowing a high fat meal.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatincrease glucose uptake in skeletal muscle cells.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatincrease glucose uptake in adipose cells.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatincrease glucose uptake in neuronal cells.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatincrease glucose uptake in red blood cells.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatincrease glucose uptake in the brain.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatsignificantly reduce the postprandial increase in plasma glucosefollowing a meal, particularly a high carbohydrate meal.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatsignificantly prevent the postprandial increase in plasma glucosefollowing a meal, particularly a high fat or a high carbohydrate meal.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatimprove insulin sensitivity.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatinhibit the progression from impaired glucose tolerance to insulinresistance.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatincrease muscle mass, preferably those that increase muscle cell number,more preferably those that increase muscle fiber number.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatpromote an increase in body girth, preferably fragments that promote anincrease in muscle mass. Further preferred OBG3 or gOBG3 polypeptidefragments promote growth rate, preferably promoting an increase ingrowth rate greater than an average growth rate in the absence of OBG3or gOBG3 polypeptide fragments.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatpromote growth rate in newborn mammals, preferably cow, goat, sheep,rabbit, mouse, rat, pig, dog, or human newborns, more preferably humannewborns between the ages of 0-6 months of age, most preferably humannewborn between the ages of 0-3 months. Further preferred OBG3 or gOBG3polypeptide fragments are those that promote growth rate in newbornunderweight or premature mammals, preferably cow, goat, sheep, rabbit,mouse, rat, pig, dog, or human underweight or premature newborns, morepreferably human underweight or premature newborns between the ages of0-6 months of age, most preferably human underweight or prematurenewborns between the ages of 0-3 months of age.

Further preferred OBG3 or gOBG3 polypeptide fragments are those thatform multimers (e.g., heteromultimers or homomultimers) in vitro and/orin vivo. Preferred multimers are homodimers or homotrimers. Otherpreferred multimers are homomultimers comprising at least 4, 6, 8, 9,10, or 12 OBG3 or gOBG3 polypeptide fragment subunits. Other preferredmultimers are heteromultimers comprising a OBG3 or gOBG3 polypeptidefragment of the invention.

Further preferred embodiments include heterologous polypeptidescomprising an OBG3 or gOBG3 polypeptide fragment of the invention.

In a second aspect, the invention features a purified, isolated, orrecombinant polynucleotide encoding said OBG3 polypeptide fragmentdescribed in the first aspect, or the complement thereof. In furtherembodiments the polynucleotides are DNA, RNA, DNA/RNA hybrids,single-stranded, and double-stranded.

In a third aspect, the invention features a recombinant vectorcomprising, consisting essentially of, or consisting of, saidpolynucleotide described in the second aspect.

In a fourth aspect, the invention features a recombinant cellcomprising, consisting essentially of, or consisting of, saidrecombinant vector described in the third aspect. A further embodimentincludes a host cell recombinant for a polynucleotide of the invention.

In a fifth aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of, said OBG3 or gOBG3 polypeptidefragment described in the first aspect and, alternatively, apharmaceutical or physiologically acceptable diluent.

In a sixth aspect, the invention features a method of reducing body masscomprising providing or administering to individuals in need of reducingbody mass said pharmaceutical or physiologically acceptable compositiondescribed in the fifth aspect. Further preferred is a method of reducingbody fat mass comprising providing or administering to individuals inneed thereof said pharmaceutical or physiologically acceptablecomposition described in the fifth aspect. Further preferred is a methodof increasing lean body mass comprising providing or administering toindividuals in need thereof said pharmaceutical or physiologicallyacceptable composition described in the fifth aspect. Further preferredis a method of increasing the growth rate of body girth or lengthcomprising providing or administering to individuals in need thereofsaid pharmaceutical or physiologically acceptable composition describedin the fifth aspect.

In preferred embodiments, the identification of said individuals in needof reducing body mass to be treated with said pharmaceutical orphysiologically acceptable composition comprises genotyping OBG3 singlenucleotide polymorphisms (SNPs) or measuring OBG3 or gOBG3 polypeptideor mRNA levels in clinical samples from said individuals. Preferably,said clinical samples are selected from the group consisting of plasma,urine, and saliva. Preferably, an OBG3 or gOBG3 polypeptide fragment ofthe present invention is administered to an individual with at least a10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in blood,serum or plasma levels of full-length OBG3 or the naturallyproteolytically cleaved OBG3 fragment as compared to healthy, non-obesepatients.

In a seventh aspect, the invention features a method of preventing ortreating an obesity-related disease or disorder comprising providing oradministering to an individual in need of such treatment saidpharmaceutical or physiologically acceptable composition described inthe fifth aspect. In preferred embodiments, the identification of saidindividuals in need of such treatment to be treated with saidpharmaceutical or physiologically acceptable composition comprisesgenotyping OBG3 single nucleotide polymorphisms (SNPs) or measuring OBG3or gOBG3 polypeptide or mRNA levels in clinical samples from saidindividuals. Preferably, said clinical samples are selected from thegroup consisting of blood, serum, plasma, urine, and saliva. Preferably,said obesity-related disease or disorder is selected from the groupconsisting of obesity, impaired glucose tolerance, insulin resistance,atherosclerosis, atheromatous disease, heart disease, hypertension,stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, orType II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or TypeI diabetes). Diabetes-related complications to be treated by the methodsof the invention include microangiopathic lesions, ocular lesions,retinopathy, neuropathy, and renal lesions. Heart disease includes, butis not limited to, cardiac insufficiency, coronary insufficiency, andhigh blood pressure. Other obesity-related disorders to be treated bycompounds of the invention include hyperlipidemia and hyperuricemia. Yetother obesity-related diseases or disorders of the invention includecachexia, wasting, AIDS-related weight loss, cancer-related weight loss,anorexia, and bulimia. In preferred embodiments, said individual is amammal, preferably a human.

In related aspects, embodiments of the present invention includesmethods of causing or inducing a desired biological response in anindividual comprising the steps of: providing or administering to anindividual a composition comprising an OBG3 or gOBG3 polypeptidefragment, wherein said biological response is selected from the groupconsisting of:

(a) lowering circulating (either blood, serum, or plasma) levels(concentration) of free fatty, acids;

(b) lowering circulating (either blood, serum or plasma) levels(concentration) of glucose;

(c) lowering circulating (either blood, serum or plasma) levels(concentration) of triglycerides:

(d) stimulating muscle lipid or free fatty acid oxidation:

(c) increasing leptin uptake in the liver or liver cells;

(e) reducing the postprandial increase in plasma free fatty acids,particularly following a high fat meal; and,

(f) reducing or eliminating ketone body production, particularlyfollowing a high fat meal;

(g) increasing tissue sensitivity to insulin, particularly muscle,adipose, liver or brain;

(h) inhibiting the progression from impaired glucose tolerance toinsulin resistance:

(i) increasing muscle cell protein synthesis;

(j) reducing adipocyte triglyceride content;

(k) increasing utilization of energy from foodstuffs or metabolicstores;

(l) increasing growth rate, preferably growth in girth or length;

(m) increasing muscle growth; and

(n) increasing skeletal growth;

and further wherein said biological response is significantly greaterthan, or at least 10%, 20%, 30%, 35%, or 40% greater than, thebiological response caused or induced by a full-length OBG3 polypeptideat the same molar concentration; or alternatively wherein saidbiological response is greater than a transient response; oralternatively wherein said biological response is sustained. In furtherpreferred embodiments, the present invention of said pharmaceutical orphysiologically acceptable composition can be used as a method tocontrol blood glucose in some persons with Noninsulin Dependent DiabetesMellitus (NIDDM. Type II diabetes) in combination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some persons with insulin DependentDiabetes Mellitus (IDDM. Type I diabetes) in combination with insulintherapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with NoninsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) in combinationwith insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) in combination with insulintherapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some persons with NoninsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, withoutcombination of insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) alone, without combination ofinsulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with NoninsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, withoutcombination of insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) alone, without combination ofinsulin therapy.

In a further preferred embodiment, the present invention may be used incomplementary therapy of NIDDM patients to improve their weight orglucose control in combination with an oral insulin secretagogue or aninsulin sensitising agent. Preferably, the oral insulin secretagogue is1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or asulphonylurea selected from tolbutamide, tolazamide, chlorpropamide,glibenclamide, glimepiride, glipizide and glidazide. Preferably, theinsulin sensitising agent is selected from metformin, ciglitazone,troglitazone and pioglitazone.

The present invention further provides a method of improving the bodyweight or glucose control of NIDDM patients alone, without an oralinsulin secretagogue or an insulin sensitising agent.

In a further preferred embodiment, the present invention may be used incomplementary therapy of IDDM patients to improve their weight orglucose control in combination with an oral insulin secretagogue or aninsulin sensitising agent. Preferably, the oral insulin secretagogue is1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or asulphonylurea selected from tolbutamide, tolazamide, chlorpropamide,glibenclamide, glimepiride, glipizide and glidazide. Preferably, theinsulin sensitising agent is selected from metformin, ciglitazone,troglitazone and pioglitazone.

The present invention further provides a method of improving the bodyweight or glucose control of IDDM patients alone, without an oralinsulin secretagogue or an insulin sensitising agent.

In a further preferred embodiment, the present invention may beadministered either concomitantly or concurrently, with the oral insulinsecretagogue or insulin sensitising agent for example in the form ofseparate dosage units to be used simultaneously, separately orsequentially (either before or after the secretagogue or either beforeor after the sensitising agent).

Accordingly, the present invention further provides for a composition ofpharmaceutical or physiologically acceptable composition and an oralinsulin secretagogue or insulin sensitising agent as a combinedpreparation for simultaneous, separate or sequential use for theimprovement of body weight or glucose control in NIDDM or IDDM patients.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition furtherprovides a method for the use as an insulin sensitiser.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some persons with NoninsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) in combinationwith insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some persons with InsulinDependent Diabetes Mellitus (IDDM, Type I diabetes) in combination withinsulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some persons with NoninsulinDependent Diabetes Mellitus (NIDDM. Type II diabetes),without insulintherapy.

In an eighth aspect, the invention features a method of making the OBG3polypeptide treatment described in the first aspect, wherein said methodis selected from the group consisting of: proteolytic cleavage,recombinant methodology and artificial synthesis.

In a ninth aspect, the present invention provides a method of making arecombinant OBG3 or gOBG3 polypeptide fragment or a full-length OBG3polypeptide, the method comprising providing a transgenic, non-humanmammal whose milk contains said recombinant OBG3 or gOBG3 polypeptidefragment or full-length protein, and purifying said recombinant OBG3 orgOBG3 polypeptide fragment or said full-length OBG3 polypeptide from themilk of said non-human mammal. In one embodiment, said non-human mammalis a cow, goat, sheep, rabbit, or mouse. In another embodiment, themethod comprises purifying a recombinant full-length OBG3 polypeptidefrom said milk, and further comprises cleaving said protein in vitro toobtain a desired OBG3 or gOBG3 polypeptide fragment.

In a tenth aspect, the invention features a use of the polypeptidedescribed in the first aspect for treatment of obesity-related diseasesand disorders and/or reducing or increasing body mass. Preferably, saidobesity-related diseases and disorders are selected from the groupconsisting of obesity, impaired glucose tolerance, insulin resistance,atherosclerosis, atherornatous disease, heart disease, hypertension,stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, orType II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or TypeI diabetes). Diabetes-related complications to be treated by the methodsof the invention include microangiopathic lesions, ocular lesions,retinopathy, neuropathy, and renal lesions. Heart disease includes, butis not limited to, cardiac insufficiency, coronary insufficiency, andhigh blood pressure. Other obesity-related disorders to be treated bycompounds of the invention include hyperlipidemia and hyperuricemia. Yetother obesity-related diseases or disorders of the invention includecachexia, wasting, AIDS-related weight loss, cancer-related weight loss,anorexia, and bulimia.

In an eleventh aspect, the invention features a use of the polypeptidedescribed in the first aspect for the preparation of a medicament forthe treatment of obesity-related diseases and disorders and/or forreducing body mass. Preferably, said obesity-related disease or disorderis selected from the group consisting of obesity, impaired glucosetolerance, insulin resistance, atherosclerosis, atheromatous disease,heart disease, hypertension, stroke. Syndrome X. Noninsulin DependentDiabetes Mellitus (NIDDM, or Type II diabetes) and Insulin DependentDiabetes Mellitus (IDDM or Type I diabetes). Diabetes-relatedcomplications to be treated by the methods of the invention includemicroangiopathic lesions, ocular lesions, retinopathy, neuropathy, andrenal lesions. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency and high blood pressure. Otherobesity-related disorders to be treated by compounds of the inventioninclude hyperlipidemia and hyperuricemia. Yet other obesity-relateddiseases or disorders of the invention include cachexia, wasting.AIDS-related weight loss, cancer-related weight loss, anorexia, andbulimia. In preferred embodiments, said individual is a mammal,preferably a human.

In a twelfth aspect, the invention provides a polypeptide of the firstaspect of the invention, or a composition of the fifth aspect of theinvention, for use in a method of treatment of the human or animal body.

In a thirteenth aspect, the invention features methods of reducing bodyweight comprising providing to an individual said pharmaceutical orphysiologically acceptable composition described in the fifth aspect, orthe polypeptide described in the first aspect. Where the reduction ofbody weight is practiced for cosmetic purposes, the individual has a BMIof at least 20 and no more than 25. In embodiments for the treatment ofobesity, the individual may have a BMI of at least 20. One embodimentfor the treatment of obesity provides for the treatment of individualswith BMI values of at least 25. Another embodiment for the treatment ofobesity provides for the treatment of individuals with BMI values of atleast 30. Yet another embodiment provides for the treatment ofindividuals with BMI values of at least 40. Alternatively, forincreasing the body weight of an individual, the BMI value should be atleast 15 and no more than 20.

In a fourteenth aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect forreducing body mass and/or for treatment or prevention of obesity-relateddiseases or disorders. Preferably, said obesity-related disease ordisorder is selected from the group consisting of obesity, impairedglucose tolerance, insulin resistance, atherosclerosis, atheromatousdisease, heart disease, hypertension, stroke, Syndrome X, NoninsulinDependent Diabetes Mellitus (NIDDM, or Type II diabetes) and InsulinDependent Diabetes Mellitus (IDDM or Type I diabetes). Diabetes-relatedcomplications to be treated by the methods of the invention includemicroangiopathic lesions, ocular lesions, retinopathy, neuropathy, andrenal lesions. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Otherobesity-related disorders to be treated by compounds of the inventioninclude hyperlipidemia and hyperuricemia. Yet other obesity-relateddiseases or disorders of the invention include cachexia, wasting,AIDS-related weight loss, cancer-related weight loss, anorexia, andbulimia. In preferred embodiments, said individual is a mammal,preferably a human. In preferred embodiments, the identification of saidindividuals to be treated with said pharmaceutical or physiologicallyacceptable composition comprises genotyping OBG3 single nucleotidepolymorphisms (SNPs) or measuring OBG3 or gOBG3 polypeptide or mRNAlevels in clinical samples from said individuals. Preferably, saidclinical samples are selected from the group consisting of blood, serum,plasma, urine, and saliva.

In a fifteenth aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect forreducing body weight for cosmetic reasons.

In a sixteenth aspect, the OBG3 or gOBG3 polypeptide fragments of theinvention features methods treating insulin resistance comprisingproviding to an individual said pharmaceutical or physiologicallyacceptable composition described in the fifth aspect, or the polypeptidedescribed in the first aspect.

In a seventeenth aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect ina method of treating individuals with normal glucose tolerance (NGT) whoare obese or who have fasting hyperinsulinemia, or who have both.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating individuals with gestationaldiabetes. Gestational diabetes refers to the development of diabetes inan individual during pregnancy, usually during the second or thirdtrimester of pregnancy.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating individuals with impairedfasting glucose (IFG). Impaired fasting glucose (IFG) is that conditionin which fasting plasma glucose levels in an individual are elevated butnot diagnostic of overt diabetes, i.e., plasma glucose levels of lessthan 126 mg/dl and greater than or equal to 110 mg/dl.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating impaired glucose tolerance(IGT) in an individual. In other further preferred embodiments, theinvention features the pharmaceutical or physiologically acceptablecomposition described in the fifth aspect in a method of preventing IGTin an individual. By providing therapeutics and methods for reducing orpreventing IGT, i.e., for normalizing insulin resistance, theprogression to NIDDM can be delayed or prevented. Furthermore, byproviding therapeutics and methods for reducing or preventing insulinresistance, the invention provides methods for reducing and/orpreventing the appearance of Insulin-Resistance Syndrome.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating a subject having polycysticovary syndrome (PCOS). PCOS is among the most common disorders ofpremenopausal women. Insulin-sensitizing agents have been shown to beeffective in PCOS. Accordingly, the invention provides methods forreducing insulin resistance, normalizing blood glucose thus treating andor preventing PCOS.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating a subject having insulinresistance. In still further preferred embodiments, a subject havinginsulin resistance is treated according to the methods of the inventionto reduce or cure the insulin-resistance. As insulin resistance is alsooften associated with infections and cancer, prevention or reducinginsulin resistance according to the methods of the invention may preventor reduce infections and cancer.

In further preferred embodiment, the methods of the invention are usedto prevent the development of insulin resistance in a subject, e.g.,those known to have an increased risk of developing insulin-resistance.

In an eighteenth aspect, the invention features a method of using OBG3or gOBG3 polypeptide fragment in a method of screening compounds for oneor more antagonists of dipeptidyl peptidase cleavage said polypeptidefragment.

In preferred embodiment, said compound is selected from but is notrestricted to small molecular weight organic or inorganic compound,protein, peptide, carbohydrate, or lipid.

In preferred embodiment, said polypeptide fragment is gOBG3 polypeptidefragment. In further preferred embodiment, said gOBG3 polypeptidefragment is gOBG3 polypeptide fragment of SEQ ID NO:6.

In preferred embodiment, said antagonist of dipeptidyl peptidasecleavage of said gOBG3 polypeptide fragment is specific for saiddipeptidyl peptidase.

In further preferred embodiment, said antagonist of dipeptidyl peptidasecleavage of said gOBG3 polypeptide fragment is specific for said gOBG3polypeptide fragment.

In a nineteenth aspect, the invention features a method of using OBG3 orgOBG3 polypeptide fragment in a method of screening compounds for one ormore antagonists of OBG3 or gOBG3 activity, wherein said activity isselected from but not restricted to lipid partitioning, lipidmetabolism, and insulin-like activity.

In preferred embodiment, said compound is selected from but is notrestricted to small molecular weight organic or inorganic compound,protein, peptide, carbohydrate, or lipid.

In a twentieth aspect, the present invention provides a mammal,preferably a newborn human, with a supplement to promote, improve,enhance or increase the assimilation, utilization or storage of energyand other nutrients present in foodstuffs consumed by newborn mammals,particularly newborn humans, and particularly energy and other nutrientsin infant formula or breast milk. A further preferred embodiment of thepresent invention is to provide a mammal, preferably a newborn human,with a supplement to promote, improve, enhance, or increase growth rate.Preferred methods of supplementation with OBG3 or gOBG3 polypeptides ofthe invention include but is not limited to:

(a) direct addition of OBG3 or gOBG3 polypeptides to synthetic infantformula or to breast milk;

(b) administration of OBG3 or gOBG3 polypeptides prior to feeding,preferably 1-15 minutes prior to feeding, more preferably 1-5 minutesprior to feeding; and

(c) administration of OBG3 or gOBG3 polypeptides following feeding,preferably 1-15 minutes following feeding, more preferably 1-5 minutesfollowing feeding;

wherein routes of administration of OBG3 or gOBG3 polypeptides areselected from oral, buccal, nasal and intramuscular routes, preferablyoral routes.

A further preferred embodiment is directed to using OBG3 or gOBG3polypeptides of the invention in methods to promote, improve, enhance orincrease the assimilation, utilization or storage of energy and othernutrients present in foodstuffs consumed by newborn mammals,particularly newborn humans, and particularly energy and other nutrientsin infant formula or breast milk. A further preferred embodiment isdirected to using OBG3 or gOBG3 polypeptides of the invention in methodsto promote, improve, enhance or increase the growth rate of a newbornmammal, preferably a human newborn. Further preferred are compositionscomprising OBG3 or gOBG3 polypeptides which can be used in methods topromote, improve, enhance or increase the assimilation, utilization orstorage of energy and other nutrients present in foodstuffs consumed bynewborn mammals, particularly newborn humans, and particularly energyand other nutrients in infant formula or breast milk. Further preferredare compositions comprising OBG3 or gOBG3 polypeptides which can be usedin methods to promote, improve, enhance or increase the growth rate of anewborn mammal, preferably a human newborn. A still further preferredembodiment of the present invention is directed to compositionscomprising synthetic infant milk formula and OBG3 or gOBG3 polypeptidesof the invention.

Another embodiment of the invention is to provide OBG3 or gOBG3polypeptide compositions useful for enhancing or improving thenutritional value of synthetic infant milk formulas or breast milk.Further preferred are compositions useful for incorporation into thediet of a newborn mammal so as to enhance and improve the nutritionalvalue of the diet. Still another embodiment of the invention is toprovide techniques and routines for improving the diet and feeding ofnewborn mammals, particularly premature, underweight orvery-low-birth-weight newborns, preferably human newborns.

In preferred embodiments of the invention, said polypeptide fragment isgOBG3 polypeptide fragment. In further preferred embodiment, said gOBG3polypeptide fragment is gOBG3 polypeptide fragment of SEQ ID NO:6.

In preferred aspects of the methods of the invention disclosed herein,the amount of OBG3 or gOBG3 polypeptide fragment or polynucleotideadministered to an individual is sufficient to bring circulating (blood,serum, or plasma) levels (concentration) of OBG3 polypeptides to theirnormal levels (levels in non-obese individuals). “Normal levels” may bespecified as the total concentration of all circulating OBG3polypeptides (full-length OBG3 and fragments thereof or theconcentration of all circulating proteolytically cleaved OBG3polypeptides only.

In preferred embodiments of the compositions of the invention disclosedherein, compositions of the invention may further comprise anycombination of OBG3 polypeptide fragments, insulin, insulinsecretagogues or insulin sensitising agents such that the compositionproduces a biological effect greater than the expected effect for anOBG3 polypeptide administered alone rather than in combination withinsulin, insulin secretagogues or insulin sensitising agents.

In a further embodiment, said biological function includes, but is notlimited to, free fatty acid level lowering activity, glucose levellowering activity, triglyceride level lowering activity, stimulatingadipose lipolysis, stimulating muscle lipid or free fatty acidoxidation, increasing leptin uptake in a liver cell line, significantlyreducing the postprandial increase in plasma free fatty acids or glucosedue to a high fat meal, significantly reducing or eliminate ketone bodyproduction as the result of a high fat meal, increasing glucose uptakein skeletal muscle cells, adipose cells, red blood cells or the brain,increasing insulin sensitivity, inhibiting the progression from impairedglucose tolerance to insulin resistance, reducing body mass, decreasingfat mass, increasing lean muscle mass, preventing or treating anmetabolic-related disease or disorder, controlling blood glucose in somepersons with Noninsulin Dependent Diabetes Mellitus or NoninsulinDependent Diabetes Mellitus, treating insulin resistance or preventingthe development of insulin resistance.

Full-length OBG3 (ACRP30. AdipoQ, APM1) polypeptides and polynucleotidesencoding the same may be specifically substituted for an OBG3 or gOBG3polypeptide fragment or polynucleotide encoding the same in anyembodiment of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of the sequences of the human (APM1), andmouse (AdipoQ and ACRP30) OBG3 polypeptides.

FIG. 2 shows the nucleic acid sequence of AdipoQ cloned into the BamHIand XhoI sites of pTrcHisB. AdipoQ begins at 510 and ends at 1214(insert in bold). This construct does not contain the N-term signalsequence (MLLLQALLFLLILP).

FIG. 3 shows a schematic drawing of the protein structure of APM1. Theputative signal sequence at the N-terminus (AA 1-17), the unique region(AA 18-41), the collagen region (AA 42-107), and the globular region (AA108-244) at the carboxy terminus are shown. Two protease cleavage sitesafter AA 100 and AA 131 are also shown.

FIG. 4 shows the nucleic acid sequence of the globular region of AdipoQcloned into pTrcHisB. AdipoQ globular region begins at 510 and ends at927 bp. The insert is in bold.

FIG. 5 is a graph showing a comparison of the effect of AdipoQ (AQ) andAdipoQ globular head (AQ-GH) on cell-associated ¹²⁵I-leptin in the mouseliver cell line BPRCL. Results are shown as percent of control values inthe presence of increasing amounts of compound (AQ or AQ-GH), and arethe mean of triplicate determinations.

FIGS. 6A, 6B, and 6C show graphs of ¹²⁵I-LDL binding, uptake, anddegradation, respectively, in the mouse liver cell line BPRCL in thepresence of increasing amounts of gOBG3.

FIG. 7 shows a protein sequence alignment of the obg3 clone (obg3 clone;the insert in FIG. 2) with the published sequences of human (APM1) andmouse (AdipoQ and ACRP30) obg3. In the alignment, amino acids (AAs) 45to 110 contain the collagen-like region; kAs 111-247 contain theglobular region. The cut sites from lysine-blocked trypsin fall afterAAs 58, 61, 95, 103, 115, 125, and 134. As determined by amino-terminalsequencing of the gOBG3 product, the gOBG3 start site is at AA 104 (101for human gOBG3 or APM1).

FIG. 8 shows a graphical representation of the effect of gOBG3 (3×25 μgip) on plasma free fatty acids (FFA) in C57BL6/J mice following a highfat meal (* p<0.02).

FIGS. 9A and 9B show graphical representations of the effect of gOBG3(3×25 μg ip) on plasma triglycerides (TG) in C57BL6/J mice following ahigh fat meal (p<0.05 at 2, 3 and 4 hours). FIG. 9A shows TG in mg/dl:FIG. 9B shows TG as a percent of the starting value.

FIG. 10 shows a graphical representation of the effect of gOBG3 (3×25 μgip) on plasma glucose in C57BL6/J mice following a high fat meal.

FIGS. 11A and 11B show graphical representations of the effect of gOBG3(3×25 μg ip) on plasma FFA in C57BL6/J mice following a high fat meal.FIG. 11A shows FFA as mM; FIG. 11B shows FFA as a percent of thestarting value.

FIGS. 12A and 12B show graphical representations of the effect of gOBG3(3×25 μg) on plasma leptin in C57BL6/J mice following a high fat meal.FIG. 12A shows leptin as ng/mL; FIG. 12B shows leptin as a percent ofthe starting value.

FIGS. 13A and 13B show graphical representations of the effect of gOBG3(3×25 μg) on plasma Insulin in C57BL6/J mice following a high fat meal.FIG. 13A shows insulin levels in ng/mL; FIG. 13B shows insulin as apercent of the starting value.

FIGS. 14A and 14B show graphical representations of the effect of OBG3or, plasma FFA in C57BL6/J mice following a high fat meal. At t=2 hoursa significant reduction in FFA was seen for both treatment groups(p<0.05). FIG. 14A shows FFA levels in mM; FIG. 14B shows FFA as apercent of the starting value.

FIGS. 15A and 15B show graphical representations of the effect of OBG3on plasma TG in C57BL6/J mice following a high fat meal. FIG. 15A showsTG levels in mg/dl; FIG. 15B shows TG as a percent of the startingvalue.

FIGS. 16A and 16B show graphical representations of the effect of OBG3on plasma glucose in C57BL6/J mice following a high fat meal. FIG. 16Ashows glucose levels as mg/dl; FIG. 16B shows glucose levels as apercent of the starting value.

FIG. 17 shows a table identifying additional APM1 SNPs. Informationconcerning Known Base Changes, Location, Prior Markers, Amplicon, andForward and Reverse primers for microsequencing are shown.

FIGS. 18A and 18B show graphical representations of the effect ofgACRP30 injection in mice on the FFA (FIG. 18A) and glucose (FIG. 18B)increases resulting from epinephrine injection.

FIG. 19 shows a graphical representation of the effect of gACRP30treatment on fatty acid metabolism in muscle isolated from mice.Treatments shown are control (white) and gACRP30 (black).

FIGS. 20A and 20B show a graphical representation of the effect ofgACRP30 treatment on triglyceride content of muscle and liver isolatedfrom mice.

FIGS. 21A, 21B, 21C, & 21D show graphical representations of the effectof gACRP30 treatment on weight gain & loss in mice. Treatments shown aresaline (diamond), ACRP30 (filled square), and gACRP30 (triangle). FIG.21A shows results of treatment of mice after 19 days on a high fat diet.FIG. 21B shows results of treatment of mice after 6 months on a high fatdiet.

FIG. 22 shows a table of the tested blood chemistry values with salineinjections, ACRP30 injections, or gACRP30 injections.

FIGS. 23A and 23B show a SDS-PAGE separation of the purification ofACRP30 and gACRP30 (23A) and a cleavage product of AMP1 (23B). FIG. 23A,Lane II shows the complete form of ACRP30 purified by FPLC. Lane 1 showsthe proteolytic cleavage product gACRP30. FIG. 23B shows a cleavageproduct of APM1 after immunoprecipitation followed by Western blotting.The apparent molecular weight of this truncated form is 27 kDa,corresponding to about 70% of the complete form of APM1 (Lane IV). Thistruncated form was not detectable when a second anti-serum, specific forthe human non-homologous region (HDQETTTQGPGVLLPLPKGA) of the proteinwas used for immunoprecipitation (Lane V) and the same anti-globularhead antiserum for detection. A preimmune serum of the same animal didnot detect any protein; a dimer of APM1 was seen with both specificantibodies (apparent MW 74 kDa).

FIG. 24 shows a graph depicting the removal of plasma FFAs afterIntralipid injection following treatment with gACRP30 (diamonds) or asaline control (triangles).

DETAILED DISCLOSURE OF THE INVENTION

Before describing the invention in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used to describe the invention herein.

As used interchangeably herein, the terms “oligonucleotides”, and“polynucleotides” and nucleic acid include RNA, DNA, or RNA/DNA hybridsequences of more than one nucleotide in either single chain or duplexform. The terms encompass “modified nucleotides” which comprise at leastone modification, including by way of example and not limitation: (a) analternative linking group, (b) an analogous form of purine, (c) ananalogous form of pyrimidine, or (d) an analogous sugar. For examples ofanalogous linking groups, purines, pyrimidines, and sugars see forexample PCT publication No. WO 95/04064. The polynucleotide sequences ofthe invention may be prepared by any known method, including synthetic,recombinant, ex vivo generation, or a combination thereof, as well asutilizing any purification methods known in the art.

The terms polynucleotide construct, recombinant polynucleotide andrecombinant polypeptide are used herein consistently with their use inthe art. The terms “upstream” and “downstream” are also used hereinconsistently with their use in the art. The terms “base paired” and“Watson & Crick base paired” are used interchangeably herein andconsistently with their use in the art. Similarly, the terms“complementary”, “complement thereof”. “complement”. “complementarypolynucleotide”. “complementary, nucleic acid” and “complementarynucleotide sequence” are used interchangeably herein and consistentlywith their use in the art.

The term “purified” is used herein to describe a polynucleotide orpolynucleotide vector of the invention that has been separated fromother compounds including, but not limited to, other nucleic acids,carbohydrates, lipids and proteins (such as the enzymes used in thesynthesis of the polynucleotide). Purified can also refer to theseparation of covalently closed polynucleotides from linearpolynucleotides, or vice versa, for example. A polynucleotide issubstantially pure when at least about 50%, 60%, 75%, or 90% of a samplecontains a single polynucleotide sequence. In some cases this involves adetermination between conformations (linear versus covalently closed). Asubstantially pure polynucleotide typically comprises about 50, 60, 70,80, 90, 95, 99% weight/weight of a nucleic acid sample. Polynucleotidepurity or homogeneity may be indicated by a number of means well knownin the art, such as agarose or polyacrylamide gel electrophoresis of asample, followed by visualizing a single polynucleotide band uponstaining the gel. For certain purposes higher resolution can be providedby using HPLC or other means well known in the art.

Similarly, the term “purified” is used herein to describe a polypeptideof the invention that has been separated from other compounds including,but not limited to, nucleic acids, lipids, carbohydrates and otherproteins. In some preferred embodiments, a polypeptide is substantiallypure when at least about 50% 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 99.5% of the polypeptide molecules of a sample have a singleamino acid sequence. In some preferred embodiments, a substantially purepolypeptide typically comprises about 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 99.5% weight/weight of a protein sample. Polypeptidepurity or homogeneity is indicated by a number of methods well known inthe art, such as agarose or polyacrylamide gel electrophoresis of asample, followed by visualizing a single polypeptide band upon stainingthe gel. For certain purposes higher resolution can be provided by usingHPLC or other methods well known in the art.

Further, as used herein, the term “purified” does not require absolutepurity; rather, it is intended as a relative definition. Purification ofstarting material or natural material to at least one order ofmagnitude, preferably two or three orders, and more preferably four orfive orders of magnitude is expressly contemplated. Alternatively,purification may be expressed as “at least” a percent purity relative toheterologous polynucleotides (DNA, RNA or both) or polypeptides. As apreferred embodiment, the polynucleotides or polypeptides of the presentinvention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99%, 99.5% or 100% pure relative to heterologouspolynucleotides or polypeptides. As a further preferred embodiment thepolynucleotides or polypeptides have an “at least” purity ranging fromany number, to the thousandth position, between 90% and 100% (e.g., atleast 99.995% pure) relative to heterologous polynucleotides orpolypeptides. Additionally, purity of the polynucleotides orpolypeptides may be expressed as a percentage (as described above)relative to all materials and compounds other than the carrier solution.Each number, to the thousandth position, may be claimed as individualspecies of purity.

The term “isolated” requires that the material be removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

Specifically excluded from the definition of “isolated” are: naturallyoccurring chromosomes (e.g., chromosome spreads), artificial chromosomelibraries, genomic libraries, and cDNA libraries that exist either as anin vitro nucleic acid preparation or as a transfected/transformed hostcell preparation, wherein the host cells are either an in vitroheterogeneous preparation or plated as a heterogeneous population ofsingle colonies. Also specifically excluded are the above librarieswherein a 5′ EST makes up less than 5% (or alternatively 1%, 2%, 3%, 4%,10%, 25%, 50%, 75%, or 90%, 95%, or 99%) of the number of nucleic acidinserts in the vector molecules. Further specifically excluded are wholecell genomic DNA or whole cell RNA preparations (including said wholecell preparations which are mechanically sheared or enzymaticallydigested). Further specifically excluded are the above whole cellpreparations as either an in vitro preparation or as a heterogeneousmixture separated by electrophoresis (including blot transfers of thesame) wherein the polynucleotide of the invention have not been furtherseparated from the heterologous polynucleotides in the electrophoresismedium (e.g., further separating by excising a single band from aheterogeneous band population in an agarose gel or nylon blot).

The term “primer” denotes a specific oligonucleotide sequence that iscomplementary to a target nucleotide sequence and used to hybridize tothe target nucleotide sequence. A primer serves as an initiation pointfor nucleotide polymerization catalyzed by DNA polymerase, RNApolymerase, or reverse transcriptase.

The term “probe” denotes a defined nucleic acid segment (or nucleotideanalog segment, e.g. PNA as defined hereinbelow) which can be used toidentify a specific polynucleotide sequence present in a sample, saidnucleic acid segment comprising a nucleotide sequence complementary tothe specific polynucleotide sequence to be identified.

The term “polypeptide” refers to a polymer of amino acids without regardto the length of the polymer. Thus, peptides, oligopeptides, andproteins are included within the definition of polypeptide. This termalso does not specify or exclude post-expression modifications ofpolypeptides. For example, polypeptides that include the covalentattachment of glycosyl groups, acetyl groups, phosphate groups, lipidgroups and the like are expressly encompassed by the term polypeptide.Also included within the definition are polypeptides which contain oneor more analogs of an amino acid (including, for example, non-naturallyoccurring amino acids, amino acids which only occur naturally in anunrelated biological system, modified amino acids from mammalian systemsetc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring. As used herein, the term “OBG3” refersgenerically to the murine or human OBG3, unless otherwise specified. Theterms “ACRP30” and “AdipoQ” refer specifically to the murine form ofOBG3 and the term “APM1” refers specifically to the human form of thegene.

Without being limited by theory, the compounds/polypeptides of theinvention are capable of modulating the partitioning of dietary lipidsbetween the liver and peripheral tissues, and are thus believed to treat“diseases involving the partitioning of dietary lipids between the liverand peripheral tissues.” The term “peripheral tissues” is meant toinclude muscle and adipose tissue. In preferred embodiments, thecompounds/polypeptides of the invention partition the dietary lipidstoward the muscle. In alternative preferred embodiments, the dietarylipids are partitioned toward the adipose tissue. In other preferredembodiments, the dietary lipids are partitioned toward the liver. In yetother preferred embodiments, the compounds/polypeptides of the inventionincrease or decrease the oxidation of dietary lipids, preferably freefatty acids (FFA) by the muscle. Dietary lipids include, but are notlimited to triglycerides and free fatty acids.

Preferred diseases believed to involve the partitioning of dietarylipids include obesity and obesity-related diseases and disorders suchas obesity, impaired glucose tolerance, insulin resistance,atherosclerosis, atheromatous disease, heart disease, hypertension,stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, orType II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or TypeI diabetes). Diabetes-related complications to be treated by the methodsof the invention include microangiopathic lesions, ocular lesions,retinopathy, neuropathy and renal lesions. Heart disease includes, butis not limited to, cardiac insufficiency, coronary insufficiency, andhigh blood pressure. Other obesity-related disorders to be treated bycompounds of the invention include hyperlipidemia and hyperuricemia. Yetother obesity-related diseases or disorders of the invention includecachexia, wasting, AIDS-related weight loss, cancer-related weight loss,anorexia, and bulimia.

The term “heterologous”, when used herein, is intended to designate anypolypeptide or polynucleotide other than an OBG3 or gOBG3 polypeptide ora polynucleotide encoding an OBG3 or gOBG3 polypeptide of the presentinvention.

The terms “comprising”, “consisting of” and “consisting essentially of”are defined according to their standard meaning. A defined meaning setforth in the M.P.E.P, controls over a defined meaning in the art and adefined meaning set forth in controlling Federal Circuit case lawcontrols over a meaning set forth in the M.P.E.P. With this in mind, theterms may be substituted for one another throughout the instantapplication in order to attach the specific meaning associated with eachterm.

The term “host cell recombinant for” a particular polynucleotide of thepresent invention, means a host cell that has been altered by the handsof man to contain said polynucleotide in a way not naturally found insaid cell. For example, said host cell may be transiently or stablytransfected or transduced with said polynucleotide of the presentinvention.

The term “obesity” as used herein is defined in the WHO classificationsof weight (Kopelman (2000) Nature 404:635-643). Underweight is less than18.5 (thin); Healthy is 18.5-24.9 (normal); grade 1 overweight is25.0-29.9 (overweight); grade 2 overweight is 30.0-39.0 (obesity); grade3 overweight is greater than or equal to 40.0 BMI. BMI is body massindex (morbid obesity) and is kg/m². Waist circumference can also beused to indicate a risk of metabolic complications where in men acircumference of greater than or equal to 94 cm indicates an increasedrisk, and greater than or equal to 102 cm indicates a substantiallyincreased risk. Similarly for women, greater than or equal to 88 cmindicates an increased risk, and greater than or equal to 88 cmindicates a substantially increased risk. The waist circumference ismeasured in cm at midpoint between lower border of ribs and upper borderof the pelvis. Other measures of obesity include, but are not limitedto, skinfold thickness which is a measurement in cm of skinfoldthickness using calipers, and bioimpedance, which is based on theprinciple that lean mass conducts current better than fat mass becauseit is primarily an electrolyte solution; measurement of resistance to aweak current (impedance) applied across extremities provides an estimateof body fat using an empirically derived equation.

The term “diabetes” as used herein is intended to encompass the usualdiagnosis of diabetes made from any of the methods included, but notlimited to, the following list: symptoms of diabetes (e.g. polyuria,polydipsia, polyphagia) plus casual plasma glucose levels of greaterthan or equal to 200 mg/dl, wherein casual plasma glucose is defined anytime of the day regardless of the timing of meal or drink consumption: 8hour fasting plasma glucose levels of less than or equal to 126 mg/dl;and plasma glucose levels of greater than or equal to 200 mg/dl 2 hoursfollowing oral administration of 75 g anhydrous glucose dissolved inwater.

The term “impaired glucose tolerance (IGT)” as used herein is intendedto indicate that condition associated with insulin-resistance that isintermediate between frank, NIDDM and normal glucose tolerance (NGT). Ahigh percentage of the IGT population is known to progress to NIDDMrelative to persons with normal glucose tolerance (Sad et al., New EnglJ Med 1988; 319:1500-6 which disclosure is hereby incorporated byreference in its entirety). Thus, by providing therapeutics and methodsfor reducing or preventing IGT, i.e., for normalizing insulinresistance, the progression to NIDDM can be delayed or prevented. IGT isdiagnosed by a procedure wherein an affected person's postprandialglucose response is determined to be abnormal as assessed by 2-hourpostprandial plasma glucose levels. In this test, a measured amount ofglucose is given to the patient and blood glucose levels measuredregular intervals, usually every half hour for the first two hours andevery hour thereafter. In a “normal” or non-IGT individual, glucoselevels rise during the first two hours to a level less than 140 mg/dland then drop rapidly. In an IGT individual, the blood glucose levelsare higher and the drop-off level is at a slower rate.

The term “Insulin-Resistance Syndrome” as used herein is intended toencompass the cluster of abnormalities resulting from an attempt tocompensate for insulin resistance that sets in motion a series of eventsthat play an important role in the development of both hypertension andcoronary artery disease (CAD), such as premature atheroscleroticvascular disease. Increased plasma triglyceride and decreasedHDL-cholesterol concentrations, conditions that are known to beassociated with CAD, have also been reported to be associated withinsulin resistance. Thus, by providing therapeutics and methods forreducing or preventing insulin resistance, the invention providesmethods for reducing and/or preventing the appearance ofinsulin-resistance syndrome.

The term “polycystic ovary syndrome (PCOS)” as used herein is intendedto designate that etiologically unassigned disorder of premenopausalwomen, affecting 5-10% of this population, characterized byhyperandrogenism, chronic anovulation, defects in insulin action,insulin secretion, ovarian steroidogenesis and fibrinolysis. Women withPCOS frequently are insulin resistant and at increased risk to developglucose intolerance or NIDDM in the third and fourth decades of life(Dunaif et al. (1996) J Clin Endocrinol Metab 81:3299 which disclosureis hereby incorporated by reference in its entirety). Hyperandrogenismalso is a feature of a variety of diverse insulin-resistant states, fromthe type A syndrome, through leprechaunism and lipoatrophic diabetes, tothe type B syndrome, when these conditions occur in premenopausal women.It has been suggested that hyperinsulinemia per se causeshyperandrogenism. Insulin-sensitizing agents, e.g., troglitazone, havebeen shown to be effective in PCOS and that, in particular, the defectsin insulin action, insulin secretion, ovarian steroidogenosis andfibrinolysis are improved (Ehrman et al. (1997) J Clin Invest 100:1230which disclosure is hereby incorporated by reference in its entirety),such as in insulin-resistant humans.

The term “insulin resistance” as used herein is intended to encompassthe usual diagnosis of insulin resistance made by any of a number ofmethods, such as the intravenous glucose tolerance test or measurementof the fasting insulin level. It is well known that there is anexcellent correlation between the height of the fasting insulin leveland the degree of insulin resistance. Therefore, one could use elevatedfasting insulin levels as a surrogate marker for insulin resistance forthe purpose of identifying which normal glucose tolerance (NGT)individuals have insulin resistance. Another way to do this is to followthe approach as disclosed in The New England Journal of Medicine, No. 3,pp. 1188 (1995) (which disclosure is hereby incorporated by reference inits entirety), i.e., to select obese subjects as an initial criterionfor entry into the treatment group. Some obese subjects have impairedglucose tolerance (IGT) while others have normal glucose tolerance(NGT). Since essentially all obese subjects are insulin resistant, i.e.,even the NGT obese subjects are insulin resistant and have fastinghyperinsulinemia. Therefore, the target of the treatment according tothe present invention can be defined as NGT individuals who are obese orwho have fasting hyperinsulinemia, or who have both.

A diagnosis of insulin resistance can also be made using the euglycemicglucose clamp test. This test involves the simultaneous administrationof a constant insulin infusion and a variable rate glucose infusion.During the test, which lasts 3-4 hours, the plasma glucose concentrationis kept constant at euglycemic levels by measuring the glucose levelevery 5-10 minutes and then adjusting the variable rate glucose infusionto keep the plasma glucose level unchanged. Under these circumstances,the rate of glucose entry into the bloodstream is equal to the overallrate of glucose disposal in the body. The difference between the rate ofglucose disposal in the basal state (no insulin infusion) and theinsulin infused state, represents insulin mediated glucose uptake. Innormal individuals, insulin causes brisk and large increase in overallbody glucose disposal, whereas in NIDDM subjects, this effect of insulinis greatly blunted, and is only 20-30% of normal. In insulin resistantsubjects with either IGT or NGT, the rate of insulin stimulated glucosedisposal is about half way between normal and NIDDM. For example, at asteady state plasma insulin concentration of about 100 μU/ml (aphysiologic level) the glucose disposal rate in normal subjects is about7 mg/kg/min. In NIDDM subjects, it is about 2.5 mg/kg/min., and inpatients with IGT (or insulin resistant subjects with NGT) it is about4-5 mg/kg/min. This is a highly reproducible and precise test, and candistinguish patients within these categories. It is also known that assubjects become more insulin resistant, the fasting insulin level rises.There is an excellent positive correlation between the height of thefasting insulin level and the magnitude of the insulin resistance asmeasured by euglycemic glucose clamp tests and, therefore, this providesthe rationale for using fasting insulin levels as a surrogate measure ofinsulin resistance.

The term “agent acting on the partitioning of dietary lipids between theliver and peripheral tissues” refers to a compound or polypeptide of theinvention that modulates the partitioning of dietary lipids between theliver and the peripheral tissues as previously described. Preferably,the agent increases or decreases the oxidation of dietary lipids,preferably free fatty acids (FFA) by the muscle. Preferably the agentdecreases or increases the body weight of individuals or is used totreat or prevent an obesity-related disease or disorder such as obesity,impaired glucose tolerance, insulin resistance, atherosclerosis,atheromatous disease, heart disease, hypertension, stroke, Syndrome X,Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) andInsulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).Diabetes-related complications to be treated by the methods of theinvention include microangiopathic lesions, ocular lesions, retinopathy,neuropathy, and renal lesions. Heart disease includes, but is notlimited to, cardiac insufficiency, coronary insufficiency, and highblood pressure. Other obesity-related disorders to be treated bycompounds of the invention include hyperlipidemia and hyperuricemia. Yetother obesity-related diseases or disorders of the invention includecachexia, wasting, AIDS-related weight loss, cancer-related weight loss,anorexia, and bulimia.

The terms “response to an agent acting on the partitioning of dietarylipids between the liver and peripheral tissues” refer to drug efficacy,including but not limited to, ability to metabolize a compound, abilityto convert a pro-drug to an active drug, and the pharmacokinetics(absorption, distribution, elimination) and the pharmacodynamics(receptor-related) of a drug in an individual.

The terms “side effects to an agent acting on the partitioning ofdietary lipids between the liver and peripheral tissues” refer toadverse effects of therapy resulting from extensions of the principalpharmacological action of the drug or to idiosyncratic adverse reactionsresulting from an interaction of the drug with unique host factors.“Side effects to an agent acting on the partitioning of dietary lipidsbetween the liver and peripheral tissues” can include, but are notlimited to adverse reactions such as dermatologic, hematologic orhepatologic toxicities and further includes gastric and intestinalulceration, disturbance in platelet function, renal injury, nephritis,vasomotor rhinitis with profuse watery secretions, angioneurotic edema,generalized urticaria, and bronchial asthma to laryngeal edema andbronchoconstriction, hypotension, and shock.

The term “OBG3-related diseases and disorders” as used herein refers toany disease or disorder comprising an aberrant functioning of OBG3, orwhich could be treated or prevented by modulating OBG3 levels oractivity. “Aberrant functioning of OBG3” includes, but is not limitedto, aberrant levels of expression of OBG3 (either increased ordecreased, but preferably decreased), aberrant activity of OBG3 (eitherincreased or decreased), and aberrant interactions with ligands orbinding partners (either increased or decreased). By “aberrant” is meanta change from the type, or level of activity seen in normal cells,tissues, or patients, or seen previously in the cell, tissue, or patientprior to the onset of the illness. In preferred embodiments, theseOBG3-related diseases and disorders include obesity and theobesity-related diseases and disorders described previously.

The term “cosmetic treatments” is meant to include treatments withcompounds or polypeptides of the invention that increase or decrease thebody mass of an individual where the individual is not clinically obeseor clinically thin. Thus, these individuals have a body mass index (BMI)below the cut-off for clinical obesity (e.g., below 25 kg/m²) and abovethe cut-off for clinical thinness (e.g., above 18.5 kg/m²). In addition,these individuals are preferably healthy (e.g., do not have anobesity-related disease or disorder of the invention). “Cosmetictreatments” ire also meant to encompass, in some circumstances, morelocalized increases in adipose tissue, for example, gains or lossesspecifically around the waist or hips, or around the hips and thighs,for example. These localized gains or losses of adipose tissue can beidentified by increases or decreases in waist or hip size, for example.

The term “preventing” as used herein refers to administering a compoundprior to the onset of clinical symptoms of a disease or condition so asto prevent a physical manifestation of aberrations associated withobesity or OBG3. Alternatively, the term “preventing” can also be usedto signify the reduction, or severity, of clinical symptoms associatedwith a disease or condition.

The term “treating” as used herein refers to administering a compoundafter the onset of clinical symptoms.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver (e.g., physician, nurse, nurse practitioner, etc in thecase of humans: veterinarian in the case of animals, including non-humanmammals) that an individual or animal requires or will benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that include the knowledgethat the individual or animal is ill, or will be ill, as the result of acondition that is treatable by the compounds of the invention.

The term “perceives a need for treatment” refers to a sub-clinicaldetermination that an individual desires to reduce weight for cosmeticreasons as discussed under “cosmetic treatment” above. The term“perceives a need for treatment” in other embodiments can refer to thedecision that an owner of an animal makes for cosmetic treatment of theanimal.

The term “individual” or “patient” as used herein refers to any animal,including mammals, preferably mice, rats, other rodents, rabbits, dogs,cats, swine, cattle, sheep, horses, or primates, and most preferablyhumans. The term may specify male or female or both, or exclude male orfemale.

The term “non-human animal” refers to any non-human vertebrate,including birds and more usually mammals, preferably primates, animalssuch as swine, goats, sheep, donkeys, horses, cats, dogs, rabbits orrodents, more preferably rats or mice. Both the terms “animal” and“mammal” expressly embrace human subjects unless preceded with the term“non-human”.

The inventors have found that a fragment of OBG3, called gOBG3, is ableto significantly reduce the postprandial response of plasma free fattyacids, glucose, and triglycerides in mice fed a high fat/sucrose meal.There was no significant effect on leptin, insulin or glucagon levels.In addition, gOBG3 was found to increase muscle free fatty acidoxidation in vitro and ex vivo. Further, gOBG3 was shown to decrease andthen to prevent an increase in weight gain in mice that had been fed ahigh fat/sucrose diet for 19 days. In mice that had been maintained onthe same high fat/sucrose diet for 6 months, gOBG3 treatment resulted ina sustained weight loss over 16 days that was significant, despite beingmaintained on the high fat/sucrose diet.

The instant invention encompasses the use of OBG3 polypeptide fragmentsin the partitioning of free fatty acid (FFA) and as an important newtool to control energy homeostasis. Of the tissues that cansignificantly remove lipids from circulation and cause FFA oxidation,muscle is quantitatively the most important. Globular OBG3 is a uniqueand novel pharmacological tool that controls body weight withoutinterfering with food intake.

PREFERRED EMBODIMENTS OF THE INVENTION I. OBG3 Polypeptide Fragments ofthe Invention

OBG3 polypeptide fragments that have measurable activity in vitro and invivo have been identified. These activities include, but are not limitedto, reduction of the postprandial response of plasma free fatty acids,glucose, and triglycerides in mice fed a high fat/sucrose meal (Example8), increase in muscle free fatty acid oxidation in vitro and ex vivo(Example 12), and sustained weight loss in mice on a high fat/sucrosediet (Example 14). Other assays for OBG3 polypeptide fragment activityin vitro and in vivo are also provided (Examples 4, 7, 9, 11, 13, forexample), and equivalent assays can be designed by those of ordinaryskill in the art.

In contrast, the “intact” or “full-length” OBG3 polypeptide does nothave either the in vivo or the in vitro activities that have beenidentified for OBG3 and gOBG3 polypeptide fragments of the invention. Inmost cases, the activities are either not present or at a minimum areundetectable over control values in the assays used. In other cases, theactivities can be measured, but are present either at extremely reducedlevels and/or require significantly more protein on a molar basiscompared with the OBG3 and gOBG3 polypeptide fragments of the invention(see, e.g. Example 10). By “intact” or “full-length” OBG3 polypeptide asused herein is meant the full-length polypeptide sequence of any OBG3polypeptide, from the N-terminal methionine to the C-terminal stopcodon. Examples of intact or full-length OBG3 polypeptides are found inSEQ ID NO:2 (mouse), SEQ ID NO:4 (mouse), and SEQ ID NO:6 (human).Theterm “OBG3 polypeptide fragments” as used herein refers to fragments ofthe “intact” or “full-length”OBG3 polypeptide that have “obesity-relatedactivity”. The term “gOBG3 polypeptide fragments” refers to polypeptidefragments comprised of part or all of the globular region and is thus anarrower term than “OBG3 polypeptide fragments”. The term “fragment”means a polypeptide having a sequence that is entirely the same as part,but not all, of an intact or full-length OBG3 polypeptide. Suchfragments may be “free-standing” (i.e., not part of or fused to otherpolypeptides), or one or more fragments may be present in a singlepolypeptide. OBG3 or gOBG3 fragments are contiguous fragments of thefull-length OBG3 polypeptide unless otherwise specified.

The term “obesity-related activity” as used herein refers to at leastone, and preferably all, of the activities described herein for OBG3polypeptide fragments. Assays for the determination of these activitiesare provided herein (e.g. Examples 4, 7-9, 11-14), and equivalent assayscan be designed by those with ordinary skill in the art. Optionally.“obesity-related activity” can be selected from the group consisting oflipid partitioning, lipid metabolism, and insulin-like activity, or anactivity within one of these categories. By “lipid partitioning”activity is meant the ability to effect the location of dietary lipidsamong the major tissue groups including, adipose tissue, liver, andmuscle. The inventors have shown that OBG3 polypeptide fragments of theinvention play a role in the partitioning of lipids to the muscle, liveror adipose tissue. By “lipid metabolism” activity is meant the abilityto influence the metabolism of lipids. The inventors have shown thatOBG3 polypeptide fragments of the invention have the ability to affectthe level of free fatty acids in the plasma as well as to increase themetabolism of lipids in the muscle through free fatty acid oxidationexperiments (Examples 4, 8, 10, 11, 12) and to transiently affect thelevels of triglycerides in the plasma and the muscle (Examples 8, 1013). By “insulin-like” activity is meant the ability of OBG3 polypeptidefragments to modulate the levels of glucose in the plasma. The inventorshave found that OBG3 polypeptide fragments do not significantly impactinsulin levels but do impact glucose levels similarly to the effects ofinsulin (Examples 9 & 10). These effects are not seen in the presence ofthe intact (full-length) OBG3 polypeptide or are significantly greaterin the presence of the OBG3 polypeptide fragments compared with thefull-length OBG3 polypeptide.

The term “significantly greater” as used herein refers to a comparisonof the activity of an OBG3 polypeptide fragment in an obesity-relatedassay compared with the activity of a full-length OBG3 polypeptide inthe same assay. By “significantly” as used herein is meant statisticallysignificant as it is typically determined by those with ordinary skillin the art. For example, data are typically calculated as a mean SEM,and a p-value≦0.05 is considered statistically significant. Statisticalanalysis is typically done using either the unpaired Student's t test orthe paired Student's t test, as appropriate in each study. Examples of asignificant change in activity as a result of the presence of an OBG3polypeptide fragment of the invention compared to the presence of afull-length OBG3 polypeptide include an increase or a decrease in agiven parameter of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, or 75%. One or more, but not necessarily all,of the measurable parameters will change significantly in the presenceof OBG3 polypeptide fragments as compared to in the presence of anintact OBG3 polypeptide.

Representative “obesity-related assays” are provided in Examples 4, 7-9,and 11-14.

These assays include, but are not limited to, methods of measuring thepostprandial response, methods of measuring free fatty acid oxidation,and methods of measuring weight modulation. In preferred embodiments,the post-prandial response is measured in non-human animals, preferablymice. In preferred embodiments changes in dietary lipids are measured,preferably free fatty acids and/or triglycerides. In other embodiments,other physiologic parameters are measured including, but not limited to,levels of glucose, insulin, and leptin. In other preferred embodiments,free fatty acid oxidation is measured in cells in vitro or ex vivo,preferably in muscle cells or tissue of non-human animals, preferablymice. In yet other preferred embodiments weight modulation is measuredin human or non-human animals, preferably rodents (rats or mice),primates, canines, felines or procines on a high fat/sucrose diet.Optionally. “obesity-related activity” includes other activities notspecifically identified herein. In general, “measurable parameters”relating to obesity and the field of metabolic research can be selectedfrom the group consisting of free fatty acid levels, free fatty acidoxidation, triglyceride levels, glucose levels, insulin levels, leptinlevels, food intake, weight, leptin and lipoprotein binding, uptake anddegradation and lipolysis stimulated receptor (LSR) expression.

In these obesity-related assays, preferred OBG3 polypeptide fragments ofthe invention, but not full-length OBG3 polypeptides, would cause asignificant change in at least one of the measurable parameters selectedfrom the group consisting of post-prandial lipemia, free fatty acidlevels, triglyceride levels, glucose levels, free fatty acid oxidation,and weight. Alternatively, preferred OBG3 poly peptide fragments of theinvention, but not full-length OBG3 polypeptides, would have asignificant change in at least one of the measurable parameters selectedfrom the group consisting of an increase in LSR activity, an increase inleptin activity and an increase in lipoprotein activity. By “LSR”activity is meant expression of LSR on the surface of the cell, or in aparticular conformation, as well as its ability to bind, uptake, anddegrade leptin and lipoprotein. By “leptin” activity is meant itsbinding, uptake and degradation by LSR, as well as its transport acrossa blood brain barrier, and potentially these occurrences where LSR isnot necessarily the mediating factor or the only mediating factor.Similarly, by “lipoprotein” activity is meant its binding, uptake anddegadation by LSR, as well as these occurrences where LSR is notnecessarily the mediating factor or the only mediating factor.

The invention is drawn, inter alia, to isolated, purified or recombinantOBG3 polypeptide fragments. OBG3 polypeptide fragments of the inventionare useful for reducing or increasing (using antagonists of OBG3polypeptides) body weight either as a cosmetic treatment or fortreatment or prevention of obesity-related diseases and disorders. OBG3polypeptide fragments are also useful inter alia in screening assays foragonists or antagonists of OBG3 fragment activity; in screening assaysfor antagonists of dipeptidyl peptidase cleavage of OBG3 fragments,preferably cleavage of the N-terminal EP dipeptide of OBG3 polypeptidefragment 103-244 of SEQ ID NO:6, cleavage of the N-terminal VP dipeptideof OBG3 fragment 85-244 of SEQ ID NO:6, or cleavage of the N-terminal EPdipeptide of OBG3 polypeptide fragment 106-247 of SEQ ID NO:2 or SEQ IDNO:4; for raising OBG3 fragment-specific antibodies: and in diagnosticassays. When used for cosmetic treatments, or for the treatment orprevention of obesity-related diseases, disorders, or conditions, one ormore OBG3 polypeptide fragments can be provided to a subject. Thus,various fragments of the full-length protein can be combined into a“cocktail” for use in the various treatment regimens.

The full-length OBG3 polypeptide is comprised of at least four distinctregions including:

1. an N-terminal putative signal sequence about from amino acids 1-17 ofSEQ ID NO:6. SEQ ID NO:2, or SEQ ID NO:4:2. a unique region about from amino acids 18-41 of SEQ ID NO:6 or 18-44of SEQ ID NO:2, or SEQ ID NO:4:3. a collagen-like region about from amino acids-2-107 of SEQ ID NO:6 or45-110 of SEQ ID NO:2 or SEQ ID NO:4; and4. a globular region about from amino acids 108-244 of SEQ ID NO:6 or111-247 of SEQ ID NO:2 or SEQ ID NO:4.

The term “collagen residues” is used in the manner standard in the artto mean the amino acid triplet glycine, X, Y, where X and Y can be anyamino acid.

The OBG3 polypeptide fragments of the present invention are preferablyprovided in an isolated form, and may be partially or substantiallypurified. A recombinantly produced version of an OBG3 polypeptidefragment can be substantially purified by the one-step method describedby Smith et al. ((1988) Gene 67(1):31-40) or by the methods describedherein or known in the art (see, e.g., Examples 1-3). Fragments of theinvention also can be purified from natural or recombinant sources usingantibodies directed against the polypeptide fragments of the inventionby methods known in the art of protein purification.

Preparations of OBG3 polypeptide fragments of the invention involving apartial purification of or selection for the OBG3 polypeptide fragmentsare also specifically contemplated. These crude preparations areenvisioned to be the result of the concentration of cells expressingOBG3 polypeptide fragments with perhaps a few additional purificationsteps, but prior to complete purification of the fragment. The cellsexpressing OBG3 polypeptide fragments are present in a pellet, they arelysed, or the crude polypeptide is lyophilized, for example.

OBG3 or gOBG3 polypeptide fragments, and polynucleotides encoding thesame, can be any integer in length from at least 6 consecutive aminoacids to 1 amino acid less than a full-length OBG3 polypeptide. Thus,for human OBG3 (SEQ ID NO:6), an OBG3 or gOBG3 polypeptide fragment canbe any integer of consecutive amino acids from 6 to 243; for mouse OBG3(SEQ ID NO:2 or SEQ ID NO:4) an OBG3 or gOBG3 fragment can be anyinteger of consecutive amino acids from 6 to 246, for example. The term“integer” is used herein in its mathematical sense and thusrepresentative integers include: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 43, 49, 50, 51, 52,53, 54, 55, 56, 57, 53, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 131, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,and 246.

Each OBG3 fragment as described above can be further specified in termsof its N-terminal and C-terminal positions. For example, everycombination of a N-terminal and C-terminal position that fragments offrom 6 contiguous amino acids to 1 amino acids less than the full-lengthOBG3 polypeptide could occupy, on any given intact and contiguousfull-length OBG3 polypeptide sequence are included in the presentinvention. Thus, a 6 consecutive amino acid fragment could occupypositions selected from the group consisting of 1-6, 2-7, 3-8, 4-9,5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20,16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30,26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40,36-41, 37-42, 38-43, 39-44, 40-45, 41-46, 42-47, 43-48, 44-49, 45-50,46-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-60,56-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70,66-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, 75-80,76-81, 77-82, 78-83, 79-84, 80-85, 81-86, 82-87, 83-88, 84-89, 85-90,86-91, 87-92, 88-93, 89-94, 90-95, 91-96, 92-97, 93-98, 94-99, 95-100,96-101, 97-102, 98-103, 99-104, 100-105, 101-106, 102-107, 103-108,104-109, 105-110, 106-111, 107-112, 108-113, 109-114, 110-115, 111-116,112-117, 113-118, 114-119, 115-120, 116-121, 117-122, 118-123, 119-124,120-125, 121-126, 122-127, 123-128, 124-129, 125-130, 126-131, 127-132,128-133, 129-134, 130-135, 131-136, 132-137, 133-138, 134-139, 135-140,136-141, 137-142, 138-143, 139-144, 140-145, 141-146, 142-147, 143-148,144-149, 145-150, 1146-151, 147-152, 148-153, 149-154, 150-155, 151-156,152-157, 153-158, 154-159, 155-160, 156-161, 157-162, 158-163, 159-164,160-165, 161-166, 162-167, 163-168, 164-169, 165-170, 166-171, 167-172,168-173, 169-174, 170-175, 171-176, 172-177, 173-178, 174-179, 175-180,176-181, 177-182, 178-183, 179-184, 180-185, 181-186, 182-187, 183-188,184-189, 185-190, 186-191, 187-192, 188-193, 139-194, 190-195, 191-196,192-197, 193-198, 194-199, 195-200, 196-201, 197-202, 198-203, 199-204,200-205, 201-206, 202-207, 203-208, 204-209, 205-210, 206-211, 207-212,208-213, 209-214, 210-215, 211-216, 212-217, 213-218, 214-219, 215-720,216-221, 217-222, 218-223, 219-224, 220-225, 221-226, 22-227,223-22.224-229, 225-230, 226-231, 227-232, 228-233, 229-234, 230-235,231-236, 232-237, 233-238, 234-239, 235-240, 236-241, 237-242, 238-243,and 239-244 of SEQ ID NO:6.

Further preferred polypeptide fragments of SEQ ID NO:6, andpolynucleotides encoding the same, are selected from the groupconsisting of fragments comprising any 50 consecutive amino acidsnumbered from 1-50, 2-51, 3-52, 4-53, 5-54, 6-55, 7-56, 8-57, 9-58,10-59, 11-60, 12-61, 13-62, 14-63, 15-64, 16-65, 17-66, 18-67, 19-68,20-69, 21-70, 22-71, 23-72, 24-73, 25-74, 26-75, 27-76, 28-77, 29-78,30-79, 31-80, 32-81, 33-82, 34-83, 35-84, 36-85, 37-86, 38-87, 39-88,40-89, 41-90, 42-91, 43-92, 44-93, 45-94, 46-95, 47-96, 48-97, 49-98,50-99, 51-100, 52-101, 53-102, 54-103, 55-104, 56-105, 57-106, 58-107,59-108, 60-109, 61-110, 62-111, 63-112, 64-113, 65-114, 66-115, 67-116,68-117, 69-118, 70-119, 71-120, 72-121, 73-122, 74-123, 75-124, 76-125,77-126, 78-127, 79-128, 80-129, 81-130, 82-131, 83-132, 84-133, 85-134,86-135, 87-136, 88-137, 89-138, 90-139, 91-140, 92-141, 93-142, 94-143,95-144, 96-145, 97-146, 98-147, 99-148, 100-149, 101-150, 102-151,103-152, 104-153, 105-154, 106-155, 107-156, 108-157, 109-158, 110-159,111-160, 112-161, 113-162, 114-163, 115-164, 116-165, 117-166, 118-167,119-168, 120-169, 121-170, 122-171, 123-172, 124-173, 125-174, 126-175,127-176, 128-177, 129-178, 130-179, 131-180, 132-181, 133-182, 134-183,135-184, 136-185, 137-186, 138-187, 139-188, 140-189, 141-190, 142-191,143-192, 144-193, 145-194, 146-195, 147-196, 148-197, 149-198, 150-199,151-200, 152-201, 153-202, 154-203, 155-204, 156-205, 157-206, 158-207,159-208, 160-209, 161-210, 162-211, 163-212, 164-213, 165-214, 166-215,167-216, 168-217, 169-218, 170-219, 171-220, 172-221, 173-222, 174-223,175-224, 176-225, 177-226, 178-227, 179-228, 180-229, 181-230, 182-231,183-232, 184-233, 185-234, 186-235, 187-236, 188-237, 189-238, 190-239,191-240, 192-241, 193-242, 194-243, 195-244 of SEQ ID NO:6.

Further preferred polypeptide fragments of SEQ ID NO:6, andpolynucleotides encoding the same, are selected from the groupconsisting of fragments comprising any 100 consecutive amino acidsnumbered from 1-100, 2-101, 3-102, 4-103, 5-104, 6-105, 7-106, 8-107,9-108, 10-109, 11-110, 12-111, 13-112, 14-113, 15-114, 16-115, 17-116,18-117, 19-118, 20-119, 21-120, 22-121, 23-122, 2-123, 25-124, 26-125,27-126, 28-127, 29-128, 30-129, 31-130, 32-131, 33-132, 34-133, 35-134,36-135, 37-136, 38-137, 39-138, 40-139, 41-140, 42-141, 43-142, 44-143,45-144, 46-145.47-146, 48-147, 49-148, 50-149, 51-150.52-151, 53-152,54-153, 55-154, 56-155, 57-156, 58-157, 59-158, 60-159, 61-160, 62-161,63-162, 64-163, 65-164, 66-165, 67-166, 68-167, 69-168, 70-169, 71-170,72-171, 73-172, 74-173.75-174, 76-175, 77-176, 78-177, 79-178, 80-179,81-180, 82-181, 83-182, 84-183, 85-184, 86-185.87-186, 88-187, 89-188,90-189, 91-190.92-191, 93-192, 94-193, 95-194, 96-195, 97-196, 98-197,99-198, 100-199, 101-200, 102-201, 103-202, 104-203, 105-204, 106-205,107-206, 108-207, 109-208, 110-209, 111-210, 112-211, 113-212, 114-213,115-214, 116-215, 117-216, 118-217, 119-218, 120-219, 121-220, 122-221,123-222, 124-223, 125-224, 126-225, 127-226, 128-227, 129-228, 130-229,131-230, 132-231, 133-232, 134-233, 135-234, 136-235, 137-236, 138-237,139-238, 140-239, 141-240, 142-241, 143-242, 144-243, and 145-244.

A 238 consecutive amino acid fragment could occupy positions selectedfrom the group consisting of 1-238, 2-239, 3-240, 4-241, 5-242, 6-243and 7-244 of SEQ ID NO:6. Similarly, the positions occupied by all theother fragments of sizes between 6 amino acids and 243 amino acids onSEQ ID NO:6 are included in the present invention and can also beimmediately envisaged based on the examples for fragments of 6, 50, 100or 238 consecutive amino acids listed above, and therefore, are notindividually listed solely for the purpose of not unnecessarilylengthening the specification. Furthermore, the positions occupied byfragments of 6 to 241 consecutive amino acids on SEQ ID NO:2 or SEQ IDNO:4 are included in the present invention and can also be immediatelyenvisaged based on these two examples and therefore are not individuallylisted solely for the purpose of not unnecessarily lengthening thespecification. In addition, the positions occupied by fragments of 6consecutive amino acids to 1 amino acid less than any other full-lengthOBG3 polypeptide can also be envisaged based on these two examples andtherefore are not individually listed solely for the purpose of notunnecessarily lengthening the specification. In preferred embodiments,OBG3 or gOBG3 polypeptide fragments, and polynucleotides encoding thesame, having unexpected activity are selected from amino acids numberedfrom 84-244, 85-244, 86-244, 87-244, 88-244, 89-244, 90-244, 91-244,92-244, 93-244, 94-244, 95-244, 96-244, 97-244, 98-244, 99-244, 100-244,101-244, 102-244, or 103-244 of SEQ ID NO:6. Still further preferred areOBG3 or gOBG3 polypeptide fragments, and polynucleotides encoding thesame, having unexpected activity, selected from amino acids numberedfrom 111-191, 144-199, or 191-244 of SEQ ID NO:6. In preferredembodiments, OBG3 or gOBG3 polypeptide fragments, and polynucleotidesencoding the same, having unexpected activity are selected from aminoacids numbered from 88-247, 89-247, 90-247, 91-247, 92-247, 93-247,94-247, 95-247, 96-247, 97-247, 98-247, 99-247, 100-247, 101-247,102-247, 103-247, 104-247, 105-247, or 106-247 of SEQ ID NO:2 or SEQ IDNO:4.

The OBG3 or gOBG3 polypeptide fragments of the present invention mayalternatively be described by the formula “n to c” (inclusive): where“n” equals the N-terminal most amino acid position (as defined by thesequence listing) and “c” equals the C-terminal most amino acid position(as defined by the sequence listing) of the polypeptide; and furtherwhere “n” equals an integer between 1 and the number of amino acids ofthe full length polypeptide sequence of the present invention minus 6(238 for SEQ ID NO: 6 and 241 for SEQ ID NOs: 2 or 4); and where “c”equals an integer between 7 and the number of amino acids of thefull-length polypeptide sequence (244 for SEQ ID NO: 6 and 247 for SEQID NOs: 2 or 4); and where “n” is an integer smaller than “c” by atleast 6. Therefore, for SEQ ID NO: 6, “n” is any integer selected fromthe list consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,230, 231, 232, 234, 235, 236, 237 and 238; and “c” is any integerselected from the group consisting of: 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244. Everycombination of “n” and “c” positions are included as specificembodiments of the invention. Moreover, the formula “n” to “c” may bemodified as ‘“n1-n2” to “c1-c2”’, wherein “n1-n2” and “c1-c2” representpositional ranges selected from any two integers above which representamino acid positions of the sequence listing. Alternative formulasinclude ‘“n1-n2” to “c”’ and ‘“n” to “c1-c2”’. In preferred embodiment,OBG3 or gOBG3 polypeptide fragments of the invention may be described bythe formula where n1=84, n2=103, and c=244 of SEQ ID NO:6 or by theformula n1=88, n2=106, and c=247 of SEQ ID NO: 2 or SEQ ID NO:4.

These specific embodiments, and other polypeptide and polynucleotidefragment embodiments described herein may be modified as being “atleast”, “equal to”, “equal to or less than”, “less than”, “at least______ but not greater than ______” or “from ______ to ______”, aspecified size or specified N-terminal and/or C-terminal positions. Itis noted that all ranges used to describe any embodiment of the presentinvention are inclusive unless specifically set forth otherwise.

The present invention also provides for the exclusion of any individualfragment specified by N-terminal and C-terminal positions or of anyfragment specified by size in amino acid residues as described above. Inaddition, any number of fragments specified by N-terminal and C-terminalpositions or by size in amino acid residues as described above may beexcluded as individual species. Further, any number of fragmentsspecified by N-terminal and C-terminal positions or by size in aminoacid residues as described above may make up a polypeptide fragment inany combination and may optionally include non-OBG3 polypeptide sequenceas well.

In particularly preferred embodiments, the OBG3 polypeptide fragment isa “globular OBG3” (gOBG3) fragment. The term “gOBG3 fragment” or “gOBG3”or “gOBG3 polypeptide” as used herein refers to fragments of afull-length OBG3 polypeptide that comprise at least 6 and any otherinteger number of amino acids up to 137 of the globular region of afull-length OBG3 polypeptide (defined above). In preferred embodiments,gOBG3 polypeptide fragments also comprise at least 1 and any otherinteger number of amino acids up 66 of the collagen region of afull-length OBG3 polypeptide, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 consecutiveamino acid residues from the collagen region of the intact OBG3polypeptide that are adjacent to the globular region. By “adjacent” tothe globular region is meant the first collagen amino acid immediatelyN-terminal to the globular region and adding each collagen amino acidconsecutively in the N-terminal direction. Thus, for example, if thereis only one collagen amino acid in the gOBG3 polypeptide fragment, it isthe collagen amino acid 107 of SEQ ID NO: 6 or amino acid 110 of SEQ IDNO:2 or SEQ ID NO:4 located adjacent and 5′ to the first amino acid ofthe globular region. If there are 24 collagen amino acids adjacent tothe globular region in the gOBG3 fragment they would be the collagenamino acids 84-107 of SEQ ID NO: 6 or amino acids 87-110 of SEQ ID NO:2or SEQ ID NO:4.

In preferred embodiments, OBG3 or gOBG3 polypeptide fragments havingunexpected activity are selected from amino acids 85-244, 86-244,87-244, 88-244, 89-244, 90-244, 91-244, 92-244, 93-244, 94-244, 95-244,96-244, 97-244.98-244, 99-244, 100-244, 101-244, 102-244, or 103-244 ofSEQ ID NO:6. In further preferred embodiments. OBG3 or gOBG3 polypeptidefragments having unexpected activity are selected from amino acids111-191, 144-199, or 191-244 of SEQ ID NO:6. In other preferredembodiments, OBG3 or gOBG3 polypeptide fragments having unexpectedactivity are selected from amino acids 88-247.89-247, 90-247, 91-247,92-247, 93-247, 94-247.95-247, 96-247.97-247, 98-247, 99-247, 100-247,101-247, 102-247, 103-247, 104-247, 105-247, or 106-247 of SEQ ID NO:2or SEQ ID NO:4. In further preferred embodiments, gOBG3 polypeptidefragments are selected from amino acids 111 to 191, 144 to 199, 191 to244, 84 to 244, 85 to 244, 101 to 244, 102 to 24, or 103 to 244 of SEQID NO:6 and amino acids 88 to 247, 104 to 247, 105 to 247, or 106 to 247of SEQ ID NO:2 or SEQ ID NO:4.

In yet other preferred embodiments, the invention features a gOBG3polypeptide fragment comprising at least 115, but not more than 175contiguous amino acids of any one of the gOBG3 fragment sequences setforth in FIG. 1, wherein no more than 24 of said at least 115 and nomore than 175 contiguous amino acids are present in the collagen-likeregion of OBG3. Preferably, the gOBG3 polypeptide fragment comprises atleast 125, but not more than 165, or at least 140, but not more than 165amino acids, and no more than 24 amino acids are in the collagen-likeregion; more preferably at least 125 but not more than 165, or at least140 but not more than 165 amino acids, and no more than 12 amino acidsare in the collagen-like region; or at least 140 and not more than 150amino acids, and no more than 8 amino acids are present in thecollagen-like region. Preferably the gOBG3 fragment is mammalian,preferably human or mouse, but most preferably human.

In further preferred embodiments of the invention, OBG3 polypeptidefragments having unexpected biological activity comprise at least 50contiguous amino acids, but no more than 137 amino acids of the globularregion of SEQ ID NO:6. More preferred are fragments comprising at least50, but not more than OBG3 and gOBG3 polypeptide fragments of theinvention include variants, fragments, analogs and derivatives of theOBG3 and gOBG3 polypeptide fragments described above, including modifiedOBG3 and gOBG3 polypeptide fragments. Particularly preferred areproteolytically cleaved fragments of OBG3 of SEQ ID NO:6, SEQ ED NO:2,or SEQ ID NO:4. More preferred is OBG3 fragment of about amino acids85-244 of SEQ ED NO:6 made by collagenase cleavage of SEQ ID NO:6 atabout position 84. More preferred is OBG3 fragment of about amino acids88-247 of SEQ ID NO:2 or of SEQ ID NO:4 made by collagenase cleavage ofSEQ ID NO:2 or SEQ ID NO:4 at about position 87. More preferred is OBG3fragment of about amino acids 85-244 of SEQ ID NO:6 made by matrixmetalloproteinase-1 (MMP-1) cleavage of SEQ ED NO:6 at about position84. More preferred is OBG3 fragment of about amino acids 88-247 of SEQID NO:2 or of SEQ ED NO:4 made by matrix metalloproteinase-1 (MMP-1)cleavage of SEQ ED NO:2 or SEQ ID NO:4 at about position 87. Morepreferred is OBG3 fragment of about amino acids 101-244 of SEQ ID NO:6made by plasmin cleavage of SEQ ID NO:6 at about position 100. Morepreferred is OBG3 fragment of about amino acids 104-247 of SEQ ID NO:2or of SEQ ED NO:4 made by plasmin cleavage of SEQ ID NO:2 or SEQ ID NO:4at about position 103. More preferred is OBG3 fragment of about aminoacids 103-244 of SEQ ID NO:6 made by precerebellin processing proteasecleavage of SEQ ID NO:6 at about position 102. More preferred is OBG3fragment of about amino acids 106-247 of SEQ ID NO:2 or of SEQ ID NO:4made by precerebellin processing protease cleavage of SEQ ED NO:2 or SEQID NO:4 at about position 105. Most preferred is APM1 proteolyticfragment of SEQ ID NO:6, wherein said APM1 fragment isolated from humanplasma migrates with an apparent molecular weight of about 27 kDa onSDS-PAGE under reducing conditions.

Variants

It will be recognized by one of ordinary skill in the art that someamino acids of the OBG3 and gOBG3 fragment sequences of the presentinvention can be varied without significant effect on the structure orfunction of the protein; there will be critical amino acids in thefragment sequence that determine activity. Thus, the invention furtherincludes variants of OBG3 and gOBG3 polypeptide fragments that haveobesity-related activity as described above. Such variants include OBG3fragment sequences with one or more amino acid deletions, insertions,inversions, repeats, and substitutions either from natural mutations orhuman manipulation selected according to general rules known in the artso as to have little effect on activity. Guidance concerning how to makephenotypically silent amino acid substitutions is provided below.

There are two main approaches for studying the tolerance of an aminoacid sequence to change (see, Bowie, et al. (1990) Science, 247,1306-10). The first method relies on the process of evolution, in whichmutations are either accepted or rejected by natural selection. Thesecond approach uses genetic engineering to introduce amino acid changesat specific positions of a cloned gene and selections or screens toidentify sequences that maintain functionality.

These studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions and indicate which amino acid changes arelikely to be permissive at a certain position of the protein. Forexample, most buried amino acid residues require nonpolar side chains,whereas few features of surface side chains are generally conserved.Other such phenotypically silent substitutions are described by Bowie etal. (supra) and the references cited therein.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala. Val, Leu and Phe;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln: exchange of the basic residues Lys and Arg: and replacements amongthe aromatic residues Phe, Tyr. In addition, the following groups ofamino acids generally represent equivalent changes: (1) Ala, Pro, Gly,Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile, Leu,Met, Ala, Phe; (4) Lys, Arg, His: (5) Phe, Tvr, Trp, His.

Similarly, amino acids in the OBG3 and gOBG3 polypeptide fragmentsequences of the invention that are essential for function can also beidentified by methods known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham, etal. (1989)

Science 244(4908):1081-5). The latter procedure introduces singlealanine mutations at every residue in the molecule. The resulting mutantmolecules are then tested for obesity-related activity using assays asdescribed above. Of special interest are substitutions of charged aminoacids with other charged or neutral amino acids that may produceproteins with highly desirable improved characteristics, such as lessaggregation. Aggregation may not only reduce activity but also beproblematic when preparing pharmaceutical or physiologically acceptableformulations, because aggregates can be immunogenic (see, e.g.,Pinckard, et al., (1967) Clin. Exp. Immunol 2:331-340; Robbins, et al.(1987) Diabetes July; 36(7):838-41; and Cleland, et al., (1993) Crit.Rev Ther Drug Carrier Syst. 10(4):307-77).

Thus, the fragment, derivative, analog, or homolog of the OBG3 or gOBG3fragment of the present invention may be, for example: (i) one in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code (i.e., may be a non-naturally occurringamino acid); or (ii) one in which one or more of the amino acid residuesincludes a substituent group: or (iii) one in which the OBG3 or gOBG3fragment is fused with another compound, such as a compound to increasethe half-life of the fragment (for example, polyethylene glycol); or(iv) one in which the additional amino acids are fused to the above formof the fragment, such as an IgG Fc fusion region peptide or leader orsecretory sequence or a sequence which is employed for purification ofthe above form of the fragment or a pro-protein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of an OBG3 or gOBG3 polypeptidefragment having an amino acid sequence which contains at least oneconservative amino acid substitution, but not more than 50 conservativeamino acid substitutions, not more than 40 conservative amino acidsubstitutions, not more than 30 conservative amino acid substitutions,and not more than 20 conservative amino acid substitutions. Alsoprovided are polypeptides which comprise the amino acid sequence of aOBG3 or gOBG3 fragment, having at least one, but not more than 10, 9, 8,7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.

Another specific embodiment of a modified OBG3 or gOBG3 fragment of theinvention is a polypeptide that is resistant to proteolysis, for examplea OBG3 or gOBG3 fragment in which a —CONH— peptide bond is modified andreplaced by one or more of the following: a (CH₂NH) reduced bond; a(NHCO) retro inverso bond; a (CH2—O) methylene-oxy bond; a (CH2—S)thiomethylene bond; a (CH2CH2) carba bond; a (CO—CH2) cetomethylenebond: a (CHOH—CH2) hydroxyethylene bond); a (N—N) bound; a E-alcenebond; or a —CH═CH— bond. Thus, the invention also encompasses an OBG3 orgOBG3 fragment or a variant thereof in which at least one peptide bondhas been modified as described above.

A further embodiment of the invention relates to an OBG3 or gOBG3polypeptide fragment made resistant to dipeptidyl peptidase cleavagethrough N-terminal modification of said polypeptide fragment. Inpreferred embodiment, said OBG3 or gOBG3 polypeptide fragment isselected from amino acids 85-244 or 103-244 of SEQ ID NO:6 or aminoacids 106-247 of SEQ ID NO:2 or SEQ ID NO:4. In preferred embodiment,said dipeptidyl peptidase cleavage leads to removal of the N-terminaldipeptide EP by dipeptidyl peptidase from said preferred gOBG3polypeptide fragment 103-244 of SEQ ID NO:6 or 106-247 of SEQ ID NO:2 orSEQ ID NO:4. In preferred embodiment, said dipeptidyl peptidase cleavageleads to removal of the N-terminal dipeptide VP by dipeptidyl peptidasefrom said preferred gOBG3 polypeptide fragment 85-244 of SEQ ID NO:6. Inpreferred embodiment, said dipeptidyl peptidase is human plasmacomprised of dipeptidyl peptidase. In preferred embodiment, saiddipeptidyl peptidase is selected from but not restricted to human CD26or human Attractin. In further preferred embodiment, said dipeptidylpeptidase is selected from soluble human CD26 or soluble humanAtrractin. In preferred embodiment, said N-terminal modification isselected from but not restricted to glycation [Harte (2001) RegulatoryPeptides 96:95-104 which disclosure is hereby incorporated by referencein its entirety]. N-methylation, alpha-methylation, desamidation[Gallwitz (2000) Regulatory Peptides 86:103-111 which disclosure ishereby incorporated by reference in its entirety], or alternation of thechirality of one or more N-terminal amino acids [Siegel (1999) EuropeanJournal of Clinical Investigation 29:610-614 which disclosure is herebyincorporated by reference in its entirety]. Thus, the invention alsoencompasses an OBG3 or gOBG3 polypeptide fragment or a variant thereofthat has been made resistant to dipeptidyl peptidase cleavage throughN-terminal modification of said polypeptide fragment.

In addition, amino acids have chirality within the body of either L orD. In some embodiments it is preferable to alter the chirality of theamino acids in the OBG3 or gOBG3 polypeptide fragments of the inventionin order to extend half-life within the body. In other embodiments, itis preferable to alter the chirality of one or more amino acid in orderto render the OBG3 or gOBG3 polypeptide fragment resistant to dipeptidylpeptidase cleavage [Siegel (1999) European Journal of ClinicalInvestigation 29:610-614 which disclosure is hereby incorporated byreference in its entirety]. In further embodiments, it is preferable toalter the chirality of the penultimate N-terminal amino acid in order torender the OBG3 or gOBG3 polypeptide fragment resistant to dipeptidylpeptidase cleavage. Thus, in some embodiments, one or more of the aminoacids are preferably in the L configuration. In other embodiments, oneor more of the amino acids are preferably in the D configuration.

Percent Identity

The polypeptides of the present invention also include polypeptideshaving an amino acid sequence at least 50% identical, at least 60%identical, or 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identical to an OBG3 or gOBG3 fragment as described above. By apolypeptide having an amino acid sequence at least, for example, 95%“identical” to an OBG3 or gOBG3 fragment amino acid sequence is meantthat the amino acid sequence is identical to the OBG3 or gOBG3polypeptide fragment sequence except that it may include up to fiveamino acid alterations per each 100 amino acids of the OBG3 or gOBG3polypeptide fragment amino acid sequence. The reference sequence is theOBG3 or gOBG3 polypeptide fragment with a sequence corresponding to thesequence of the sequence listing. Thus, to obtain a polypeptide havingan amino acid sequence at least 95% identical to an OBG3 or gOBG3fragment amino acid sequence, up to 5% (5 of 100) of the amino acidresidues in the sequence may be inserted, deleted, or substituted withanother amino acid compared with the OBG3 or gOBG3 polypeptide fragmentsequence. These alterations may occur at the amino or carboxy termini oranywhere between those terminal positions, interspersed eitherindividually among residues in the sequence or in one or more contiguousgroups within the sequence.

As a practical matter, whether any particular polypeptide is apercentage identical to an OBG3 or gOBG3 fragment can be determinedconventionally using known computer programs. Such algorithms andprograms include, but are by no means limited to, TBLASTN, BLASTP,FASTA, TFASTA, and CLUSTALW (Pearson and Lipman. (1988) Proc Natl AcadSci USA 85(8):2444-8: Altschul et al. (1990) J Mol Biol 215(3):403-410;Thompson et al., (1994) Nucleic Acids Res 22(2):4673-4680: Higgins etal. (1996) Meth Enzymol 266:383-402: Altschul et al., (1997) Nuc AcidsRes 25:3389-3402; Altschul et al. (1993) Nature Genetics 3:266-272). Ina particularly preferred embodiment, protein and nucleic acid sequencehomologies are evaluated using the Basic Local Alignment Search Tool(“BLAST”), which is well known in the art (See, e.g. Karlin and Altschul(1990) Proc Natl Acad Sci USA 87(6):226-8: Altschul et al. 1990, 1993,1997, all supra). In particular, five specific BLAST programs are usedto perform the following tasks:

(1) BLASTP and BLAST3 compare an amino acid query sequence against aprotein sequence database:

(2) BLASTN compares a nucleotide query sequence against a nucleotidesequence database;

(3) BLASTX compares the six-frame conceptual translation products of aquery nucleotide sequence (both strands) against a protein sequencedatabase;

(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and

(5) TBLASTX compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (i.e., aligned) bymeans of a scoring matrix, many of which are known in the art.Preferably, the scoring matrix used is the BLOSUM62 matrix (see, Gonnetet al., (1992) Science 256(5062):1443-5; Henikoff and Henikoff (1993)Proteins 17(1):49-61). Less preferably, the PAM or PAbM250 matrices mayalso be used (See, e.g., Schwartz and Dayhoff, eds. (1978) Matrices forDetecting Distance Relationships: Atlas of Protein Sequence andStructure, Washington: National Biomedical Research Foundation). TheBLAST programs evaluate the statistical significance of all high-scoringsegment pairs identified, and preferably selects those segments whichsatisfy a user-specified threshold of significance, such as auser-specified percent homology. Preferably, the statisticalsignificance of a high-scoring segment pair is evaluated using thestatistical significance formula of Karlin (See, e.g., Karlin andAltschul, (1990) Proc Natl Acad Sci USA 87(6):2264-8). The BLASTprograms may be used with the default parameters or with modifiedparameters provided by the user. Preferably, the parameters are defaultparameters.

A preferred method for determining the best overall match between aquery sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, can bedetermined using the FASTDB computer program based on the algorithm ofBrutlag et al. (1990) Comp. App. Biosci. 6:237-245. In a sequencealignment the query and subject sequences are both amino acid sequences.The result of said global sequence alignment is in percent identity.Preferred parameters used in a FASTDB amino acid alignment are:Matrix=PAM 0, k-tuple=2. Mismatch Penalry=1 Joining Penalty=20,Randomization Group=25 Length=0, Cutoff Score=1, Window Size=sequencelength. Gap Penalty—5, Gap Size Penalty=0.05, Window Size=247 or thelength of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, the results, inpercent identity, must be manually corrected because the FASTDB programdoes not account for N- and C-terminal truncations of the subjectsequence when calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal of the subject sequence,that are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. Whether a residue ismatched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This final percent identityscore is what is used for the purposes of the present invention. Onlyresidues to the N- and C-termini of the subject sequence, which are notmatched/aligned with the query sequence, are considered for the purposesof manually adjusting the percent identity score. That is, only queryamino acid residues outside the farthest N- and C-terminal residues ofthe subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100-residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not match/align with the first residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.

In another example, a 90-residue subject sequence is compared with a100-residue query sequence. This time the deletions are internal sothere are no residues at the N- or C-termini of the subject sequence,which are not matched/aligned with the query. In this case, the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected. No othermanual corrections are made for the purposes of the present invention.

Production

Note, throughout the disclosure, wherever OBG3 polypeptide fragments arediscussed, gOBG3 fragments are specifically intended to be included as apreferred subset of OBG3 polypeptide fragments.

OBG3 polypeptide fragments are preferably isolated from human ormammalian tissue samples or expressed from human or mammalian genes inhuman or mammalian cells. The OBG3 polypeptide fragments of theinvention can be made using routine expression methods known in the art.The polynucleotide encoding the desired polypeptide fragments is ligatedinto an expression vector suitable for any convenient host. Botheukaryotic and prokaryotic host systems are used in forming recombinantpolypeptide fragments. The polypeptide fragment is then isolated fromlysed cells or from the culture medium and purified to the extent neededfor its intended use. Purification is by any technique known in the art,for example, differential extraction, salt fractionation,chromatography, centrifugation, and the like. See, for example, Methodsin Enzymology for a variety of methods for purifying proteins. Also, seeExamples 1-3 for methods previously used for OBG3 polypeptide fragments.

In an alternative embodiment, the polypeptides of the invention areisolated from milk. The polypeptides can be purified as full-length OBG3polypeptides, which can then be cleaved, if appropriate, in vitro togenerate an OBG3 fragment, or, alternatively, OBG3 fragments themselvescan be purified from the milk. Any of a large number of methods can beused to purify the present polypeptides from milk, including thosetaught in Protein Purification Applications. A Practical Approach (NewEdition), Edited by Simon Roe, AEA Technology Products and Systems.Biosciences, Harwell; Clark (1998) J Mammary Gland Biol Neoplasia3:337-50, Wilkins and Velander (1992) 49:333-8: U.S. Pat. Nos.6,140,552; 6,025,540; Hennighausen, Protein Expression and Purification,vol. 1, pp. 3-8 (1990) Harris et al. (1997) Bioseparation 7:31-7:Degener et al. (1998) J Chromatog 799:125-37; Wilkins (1993) J CellBiochem Suppl. 0 (17 part A):39; the entire disclosures of each of whichare herein incorporated by reference. In a typical embodiment, milk iscentrifuged, e.g., at a relatively low speed, to separate the lipidfraction, and the aqueous supernatant is then centrifuged at a higherspeed to separate the casein in the milk from the remaining, “whey”fraction. Often, biomedical proteins are found in this whey fraction,and can be isolated from this fraction using standard chromatogaphic orother procedures commonly used for protein purification, e.g., asdescribed elsewhere in the present application. In one preferredembodiment, OBG3 polypeptides are purified using antibodies specific toOBG3 polypeptides, e.g., using affinity chromatography. In addition,methods can be used to isolate particular OBG3 fragments, e.g.,electrophoretic or other methods for isolating proteins of a particularsize. The OBG3 polypeptides isolating using these methods can benaturally occurring, as OBG3 polypeptides have been discovered to benaturally present in the milk of mammals (see, e.g. Example 17), or canbe the result of the recombinant production of the protein in themammary glands of a non-human mammal, as described infra, in one suchembodiment, the OBG3 fragment is produced as a fusion protein with aheterologous, antigenic polypeptide sequence, which antigenic sequencecan be used to purify the protein, e.g., using standard immuno-affinitymethodology.

In addition, shorter protein fragments may be produced by chemicalsynthesis. Alternatively, the proteins of the invention are extractedfrom cells or tissues of humans or non-human animals. Methods forpurifying proteins are known in the art, and include the use ofdetergents or chaotropic agents to disrupt particles followed bydifferential extraction and separation of the polypeptides by ionexchange chromatography, affinity chromatography, sedimentationaccording to density, and gel electrophoresis.

Any OBG3 fragment cDNA, including that in FIG. 4, can be used to expressOBG3 polypeptide fragments. The nucleic acid encoding the OBG3 fragmentto be expressed is operably linked to a promoter in an expression vectorusing conventional cloning technology. The OBG3 fragment cDNA insert inthe expression vector may comprise the coding sequence for: thefull-length OBG3 polypeptide (to be later modified); from 6 amino acidsto 6 amino acids less than the full-length OBG3 polypeptide; a gOBG3fragment; or variants and % similar polypeptides.

The expression vector is any of the mammalian, yeast, insect orbacterial expression systems known in the art, some of which aredescribed herein, and examples of which are given in the Examples(Examples 1-3). Commercially available vectors and expression systemsare available from a variety of suppliers including Genetics Institute(Cambridge, L4), Stratagene (La Jolla, Calif.), Promega (Madison, Wis.),and Invitrogen (San Diego, Calif.). If desired, to enhance expressionand facilitate proper protein folding, the codon context and codonpairing of the sequence can be optimized for the particular expressionorganism into which the expression vector is introduced, as explained byHatfield, et al. U.S. Pat. No. 5,082,767, the disclosures of which areincorporated by reference herein in their entirety.

If the nucleic acid encoding OBG3 polypeptide fragments lacks amethionine to serve as the initiation site, an initiating methionine canbe introduced next to the first codon of the nucleic acid usingconventional techniques. Similarly, if the insert from the OBG3polypeptide fragment cDNA lacks a poly A signal, this sequence can beadded to the construct by, for example, splicing out the Poly A signalfrom pSG5 (Stratagene) using BglI and SalI restriction endonucleaseenzymes and incorporating it into the mammalian expression vector pXT1(Stratagene), pXT1 contains the LTRs and a portion of the gag gene fromMoloney Murine Leukemia Virus. The position of the LTRs in the constructallow efficient stable transfection. The vector includes the HerpesSimplex Thymidine Kinase promoter and the selectable neomycin gene.

The nucleic acid encoding an OBG3 fragment can be obtained by PCR from avector containing the OBG3 nucleotide sequence using oligonucleotideprimers complementary to the desired OBG3 cDNA and containingrestriction endonuclease sequences for Pst I incorporated into the 5′primer and BglII at the 5′ end of the corresponding cDNA 3′ primer,taking care to ensure that the sequence encoding the OBG3 fragment ispositioned properly with respect to the poly A signal. The purifiedfragment obtained from the resulting PCR reaction is digested with PstI,blunt ended with an exonuclease, digested with Bgl II, purified andligated to pXT1, now containing a poly A signal and digested with BglII.Alternative methods are presented in Examples 1-3.

Transfection of an OBG3 fragment-expressing vector into mouse NIH 3T3cells is one embodiment of introducing polynucleotides into host cells.Introduction of a polynucleotide encoding a polypeptide into a host cellcan be effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection, or other methods. Such methods are described inmany standard laboratory manuals, such as Davis et al. ((1986) Methodsin Molecular Biology, Elsevier Science Publishing Co., Inc., Amsterdam).It is specifically contemplated that the polypeptides of the presentinvention may in fact be expressed by a host cell lacking a recombinantvector. Methods of expressing OBG3 fragment of the invention in cellsare described in Examples 1-3.

A polypeptide of this invention (i.e., an OBG3 or gOBG3 fragment) can berecovered and purified from recombinant cell cultures by well-knownmethods including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxyylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Polypeptides of the presentinvention, and preferably the secreted form, can also be recovered from:products purified from natural sources, including bodily fluids, tissuesand cells, whether directly isolated or cultured; products of chemicalsynthetic procedures: and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect, and mammalian cells.

Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated.

Preferably the polypeptides of the invention are non-glycosylated. Inaddition, polypeptides of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins, thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with the polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences via homologous recombination, see,e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication No. WO 96/29411, published Sep. 26, 1996: InternationalPublication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,(1989) Proc Natl Acad Sci USA 86(22):8932-5; Koller et al., (1989) ProcNatl Acad Sci USA 86(22):8927-31; and Zijlstra et al. (1989) Nature342(6248):435-8; the disclosures of each of which are incorporated byreference in their entireties).

Modifications

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (See, e.g. Creighton. 1983 Proteins.New York, N.Y.: W.H. Freeman and Company; and Hunkapiller et al., (1984)Nature 310(5973):105-11). For example, a relative short fragment of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thefragment sequence. Non-classical amino acids include, but are norlimited to, to the D-isomers of the common amino acids.2,4-diaminobutyric acid. α-amino isobutyric acid, 4-aminobutyric acid.Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid. Aib.2-amino isobutyric acid. 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acidssuch as b-methyl amino acids. Ca-methyl amino acids. Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptide fragments which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited, to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain. VS protease. NaBH₄; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptide fragments may also be modified with adetectable label, such as an enzymatic, fluorescent, isotopic oraffinity label to allow for detection and isolation of the polypeptide.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention that may provide additional advantagessuch as increased solubility, stability and circulating time of thepolypeptide, or decreased immunogenicity. See U.S. Pat. No. 4,179,337.The chemical moieties for derivitization may be selected from watersoluble polymers such as polyethylene glycol, ethylene glycol/propyleneglycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcoholand the like. The polypeptides may be modified at random positionswithin the molecule, or at predetermined positions within the moleculeand may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the polypeptide with consideration of effects on functionalor antigenic domains of the polypeptide. There are a number ofattachment methods available to those skilled in the art, e.g. EP 0 401384, herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al. (1992) Exp Hematol 20(8):1028-35, reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues: those having a free carboxyl group mayinclude aspartic acid residues, glutamic acid residues and theC-terminal amino acid residue. Sulfhydryl groups may also be used as areactive group for attaching the polyethylene glycol molecules.Preferred for therapeutic purposes is attachment at an amino group, suchas attachment at the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus may be accomplished by reductive alkylation,which exploits differential reactivity of different types of primaryamino groups (lysine versus the N-terminal) available for derivatizationin a particular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved.

In further preferred embodiment, the invention features a method ofreducing body mass comprising providing or administering to individualsin need of reducing body mass said pharmaceutical or physiologicallyacceptable composition described in the fifth aspect in combination withprovision or administration of an antagonist of dipeptidyl peptidasecleavage of OBG3 or gOBG3 polypeptide fragment of the first aspect.

Preferred said antagonist is a peptidyl derivative of a diester ofalpha-aminoalkylphosphonic acid (U.S. Pat. No. 5,543,396 whichdisclosure is hereby incorporated by reference in its entirety). Morepreferred said peptidyl derivative is selected from Ala-Pro^(P)(OZ)₂.AcOH.Ala-Pip^(P) (Oph)₂, HCl.Ala-Pro^(P) (Oph-4C)₂,HCl.Ala-Pip^(P)(Oph-4Cl)₂, or 2HCl.Lys-Pip^(P) (Oph-4Cl)₂, where Zrepresents an aryl group, a substituted aryl group or a highlyflourinated alkyl group. Pro^(p) represents a proline phosphonatederivative, and Pip^(P) represents piperidyl phosphonate (U.S. Pat. No.5,542,396 which disclosure is hereby incorporated by reference in itsentirety).

Other preferred said antagonist is a compound of the general formulaZ-Xaa-Y′, in which Xaa is an amino acid, Z is a protecting group, and Y′is one of various types of ring structures (U.S. Pat. No. 6,090,786which disclosure is hereby incorporated by reference in its entirety).More preferred is said compound wherein Z may or may not be present andrepresents a protecting group, such as benzyloxycarbonyl; Xaa representsalanine, methionine, arginine, phenylalanine, aspartic acid, proline,asparagine, serine, cysteine, threonine, glycine, tyrosine, glutamicacid, tryptophan, glutamine, valine, isoleucine, lysine, leucine,L-thioproline, L-homoproline,L-1,2,3,4,tetrahydroisoquinoline-3-carboxylic acid (Tic).L-2,3-dihydroindol-2-carboxylic acid, L-naphthylglycine,L-phenylglycine, L-4-phenylproline, O-benzyl tyrosine, omega-Z lysine,or omega-acetyl lysine; and Y′ represents a pyrrolidide, a phosphonateor phosphinate derivative, or reduced peptide; or pharmaceuticallyacceptable salts thereof (U.S. Pat. No. 6,090,786 which disclosure ishereby incorporated by reference in its entirety).

Other preferred said antagonist is sulphostin (U.S. Pat. No. 6,214,340which disclosure is hereby incorporated by reference in its entirety).

Other preferred said antagonist is N-(substitutedglycyl)-2-cyanopyrrolidine (U.S. Pat. No. 6,166,063). More preferredsaid N-(substituted glycyl)-2-cyanopyrrolidine is selected frompyrrolidine, 1-[[(3,5-dimethyl-1-adamantyl)amino]-aceryl]-2-cyano-,(S)—; pyrrolidine, 1-[[(3-ethyl-1-adamantyl)amino]-aceryl]-2-cyano-,(S)—; pyrrolidine, 1-[[(3-methoxy-1-adamantyl)amino]-acetyl]-2-cyano-,(S)—; pyrrolidine,1-[[[3-[[(t-butylamino)carbonyl]oxy]-1-adamantyl]amino]-acetyl]-2-cyano-,(S)—; pyrrolidine,1-[[[3-[[[(4-methoxyphenyl)amino]-carbonyl]oxy]-1-adamanwyl]amino]-acetyl]-2-cyano-,(S)—: pyrrolidine,1-[[[(3-[[(phenylamino)carbonyl]oxy]-1-adamantyl]amino]-acetyl]-2-cyano-,(S)—: pyrrolidine, 1-[[(5-hydroxy-2-adamantyl))amino]-acetyl]-2-cyano-,(S)—; pyrrolidine, 1-[[(3-acetyloxy-1-adamantyl)amino]-acetyl]-2-cyano-,(S)—; pyrrolidine,1-[[[(3-[[[(diisopropyl)amino]carbonyl]oxy]-1-adamantyl]amino]-acetyl]-2-cyano-,(S)—: pyrrolidine,1-[[[3-[[[(cyclohexyl)amino]carbonyl]oxy]-1-adamantyl]amino]-acetyl]-2-cyano-,(S)—: pyrrolidine, 1-[[(3-ethyoxy-1-adamantyl)amino]-acetyl]-2-cyano-,(S)—: or, in each case, a pharmaceutically acceptable acid addition saltthereof (U.S. Pat. No. 6,166,063).

Other preferred said antagonist is terrahydroisoqumoline 3-carboxamidederivative of formula ##STR1## (U.S. Pat. No. 6,172,081). More preferredis said derivative and pharmaceutically acceptable salts thereof whereinN is CH₂, S, O, or C(CH₃)₂; R₁ and R₂ are independently hydrogen,hydroxy, alkyl, alkoxy, aralkoxy, or halogen (U.S. Pat. No. 6,172,081).

Other preferred said antagonist is valine-pyrrolidide [Deacon (2001)Diabetes 50:1588-1597 which disclosure is hereby incorporated byreference in its entirety].

Multimers

The polypeptide fragments of the invention may be in monomers ormultimers (i.e., dimers, trimers, tetramers and higher multimers).Accordingly, the present invention relates to monomers and multimers ofthe polypeptide fragments of the invention, their preparation, andcompositions (preferably, pharmaceutical or physiologically acceptablecompositions) containing them. In specific embodiments, the polypeptidesof the invention are monomers, dimers, trimers or tetramers. Inadditional embodiments, the multimers of the invention are at leastdimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlypolypeptides corresponding to the OBG3 polypeptide fragments of theinvention (including polypeptide fragments, variants, splice variants,and fusion proteins corresponding to these polypeptide fragments asdescribed herein). These homomers may contain polypeptide fragmentshaving identical or different amino acid sequences. In a specificembodiment, a homomer of the invention is a multimer containing onlypolypeptide fragments having an identical amino acid sequence. Inanother specific embodiment, a homomer of the invention is a multimercontaining polypeptide fragments having different amino acid sequences.In specific embodiments, the multimer of the invention is a homodimer(e.g., containing polypeptide fragments having identical or differentamino acid sequences) or a homotrimer (e.g., containing polypeptidefragments having identical and/or different amino acid sequences). Inadditional embodiments, the homomeric multimer of the invention is atleast a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., corresponding to differentproteins or polypeptide fragments thereof) in addition to thepolypeptides of the invention. In a specific embodiment, the multimer ofthe invention is a heterodimer, a heterotrimer, or a heterotetramer. Inadditional embodiments, the heteromeric multimer of the invention is atleast a heterodimer, at least a heterotrimer, or at least aheterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences,which interact in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

In one example, covalent associations are between the heterologoussequence contained in a fusion protein of the invention (see, e.g., U.S.Pat. No. 5,478,925). In a specific example, the covalent associationsare between the heterologous sequence contained in an Fc fusion proteinof the invention (as described herein). In another specific example,covalent associations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, such as for example,oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins, and have since been found ina variety of different proteins (Landschulz et al. (1988) Genes Dev.July: 2(7):786-800). Among the known leucine zippers are naturallyoccurring peptides and derivatives thereof that dimerize or trimerize.Examples of leucine zipper domains suitable for producing solublemultimeric proteins of the invention are those described in PCTapplication WO 94/10308, hereby incorporated by reference. Recombinantfusion proteins comprising a polypeptide of the invention fused to apolypeptide sequence that dimerizes or trimerizes in solution areexpressed in suitable host cells, and the resulting soluble multimericfusion protein is recovered from the culture supernatant usingtechniques known in the art.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. FEBS Letters (1994) 344(2-3): 191-5and in U.S. patent application Ser. No. 08/446,922, hereby incorporatedby reference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention. In another example, proteins of the invention are associatedby interactions between Flag® & polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, proteins of the invention are associated byinteractions between heterologous polypeptide sequence contained inFlag® fusion proteins of the invention and anti Flag® antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g. U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C-terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, at least 30techniques known in the art may be applied to generate liposomescontaining the polypeptide components desired to be contained in themultimer of the invention (see, e.g. U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g. U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety. In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hyrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (See, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

II. OBG3 Polynucleotides of the Invention

Preferred polynucleotides are those that encode OBG3 and gOBG3polypeptide fragments of the invention. The recombinant polynucleotidesencoding OBG3 and gOBG3 polypeptide fragments can be used in a varietyof ways, including, but not limited to, expressing the polypeptide inrecombinant cells for use in screening assays for antagonists andagonists of its activity as well as to facilitate its purification foruse in a variety of ways including, but not limited to screening assaysfor agonists and antagonists of its activity, diagnostic screens, andraising antibodies, as well as treatment and/or prevention ofobesity-related diseases and disorders and/or to reduce body mass.

The invention relates to the polynucleotides encoding OBG3 and gOBG3polypeptide fragments and variant polypeptide fragments thereof asdescribed herein. These polynucleotides may be purified, isolated,and/or recombinant. In all cases, the desired OBG3 and gOBG3polynucleotides of the invention are those that encode OBG3 and gOBG3polypeptide fragments of the invention have obesity-related activity asdescribed and discussed herein.

Fragments

A polynucleotide fragment is a polynucleotide having a sequence thatentirely is the same as part, but not all, of the full-length OBG3polypeptide or a specified OBG3 or gOBG3 polypeptide nucleotidesequence. Such fragments may be “free-standing”, i.e., not part of orfused to other polynucleotides, or they may be comprised within anothernon-OBG3 or non-gOBG3 (heterologous) polynucleotide of which they form apart or region. However, several OBG3 or gOBG3 polynucleotide fragmentsmay be comprised within a single polynucleotide.

The OBG3 polynucleotides of the invention comprise from 18 consecutivebases to 18 consecutive bases less than the full-length polynucleotidesequence encoding the intact OBG3 polypeptide, for example thefull-length OBG3 polypeptide polynucleotide sequences in SEQ ID NO: 1,SEQ ID NO:3, or SEQ ID NO:5. In one aspect of this embodiment, thepolynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135,140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205,210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275,280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345,350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415,420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485,490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555,560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625,630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695,700, 705, 710, 715, 720, 725, or 740 consecutive nucleotides of apolynucleotide of the present invention.

In addition to the above preferred nucleic acid sizes, further preferrednucleic acids comprise at least 18 nucleotides, wherein “at least 18” isdefined as any integer between 18 and the integer representing 18nucleotides less than the 3′ most nucleotide position of the intact OBG3polypeptide cDNA as set forth in the sequence listing (SEQ ID NO: 1, SEQID NO:3, or SEQ ID NO:5) or elsewhere herein.

Further included as preferred polynucleotides of the present inventionare nucleic acid fragments at least 18 nucleotides in length, asdescribed above, that are further specified in terms of their 5′ and 3′position. The 5′ and 3′ positions are represented by the positionnumbers set forth in the sequence listing below. For allelic anddegenerate and other variants, position 1 is defined as the 5′ mostnucleotide of the ORF, i.e., the nucleotide “A” of the start codon (ATG)with the remaining nucleotides numbered consecutively. Therefore, everycombination of a 5′ and 3′ nucleotide position that a polynucleotidefragment invention, at least 18 contiguous nucleotides in length, couldoccupy on an intact OBG3 polypeptide polynucleotide of the presentinvention is included in the invention as an individual species. Thepolynucleotide fragments specified by 5′ and 3′ positions can beimmediately envisaged and are therefore not individually listed solelyfor the purpose of not unnecessarily lengthening the specification.

It is noted that the above species of polynucleotide fragments of thepresent invention may alternatively be described by the formula “x toy”; where “x” equals the 5′ most nucleotide position and “y” equals the3′ most nucleotide position of the polynucleotide: and further where “x”equals an integer between 1 and the number of nucleotides of thepolynucleotide sequence of the present invention minus 18, and where “y”equals an integer between 19 and the number of nucleotides of thepolynucleotide sequence of the present invention minus 18 nucleotides:and where “x” is an integer smaller then “v” by at least 18.

The present invention also provides for the exclusion of any species ofpolynucleotide fragments of the present invention specified by 5′ and 3′positions or polynucleotides specified by size in nucleotides asdescribed above. Any number of fragments specified by 5′ and 3′positions or by size in nucleotides, as described above, may beexcluded.

The gOBG3 polynucleotide fragments of the invention comprise from 18consecutive bases to the full-length polynucleotide sequence encodingthe gOBG3 fragments described in Section II of the Preferred Embodimentsof the Invention. In one aspect of this embodiment, the polynucleotidecomprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290,295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360,365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430,435, 440, 445, 450, 455, 460, or 465 consecutive nucleotides of apolynucleotide of the present invention.

In addition to the above preferred nucleic acid sizes, further preferrednucleic acids comprise at least 18 nucleotides, wherein “at least 18” isdefined as any integer between 18 and the integer corresponding to the3′ most nucleotide position of a gOBG3 fragment cDNA herein.

Further included as preferred polynucleotides of the present inventionare nucleic acid fragments at least 18 nucleotides in length, asdescribed above, that are further specified in terms of their 5′ and 3′position. The 5′ and 3′ positions are represented by the positionnumbers set forth in the sequence listing below. For allelic anddegenerate and other variants, position 1 is defined as the 5′ mostnucleotide of the open reading frame (ORF), i.e., the nucleotide “A” ofthe start codon (ATG) with the remaining nucleotides numberedconsecutively. Therefore, every combination of a 5′ and 3′ nucleotideposition that a polynucleotide fragment invention, at least 18contiguous nucleotides in length, could occupy on a gOBG3 fragmentpolynucleotide of the present invention is included in the invention asan individual species. The polynucleotide fragments specified by 5′ and3′ positions can be immediately envisaged and are therefore notindividually listed solely for the purpose of not unnecessarilylengthening the specification.

It is noted that the above species of polynucleotide fragments of thepresent invention may alternatively be described by the formula “x toy”: where “x” equals the 5′ most nucleotide position and “y” equals the3′ most nucleotide position of the polynucleotide: and further where “x”equals an integer between 1 and the number of nucleotides of the gOBG3polynucleotide sequence of the present invention minus 18, and where “y”equals an integer between 9 and the number of nucleotides of the gOBG3polynucleotide sequence of the present invention: and where “x” is aninteger smaller than “y” by at least 18. Every combination of “x” and“y” positions are included as specific embodiments of the invention.Moreover, the formula “x” to “y” may be modified as ‘“x1-x2” to“y1-y2”’, wherein “x1-x2” and “y1-y2” represent positional rangesselected from any two nucleotide positions of the sequence listing.Alternative formulas include ‘“x1-x2” to “y”’ and ‘“x” to “y1-y2”’.

These specific embodiments, and other polynucleotide fragmentembodiments described herein may be modified as being “at least”, “equalto”, “equal to or less than”, “less than”, “at least ______ but notgreater than ______” or “from ______ to ______”, a specified size orspecified 5′ and/or 3′ positions.

The present invention also provides for the exclusion of any species ofpolynucleotide fragments of the present invention specified by 5′ and 3′positions or polynucleotides specified by size in nucleotides asdescribed above. Any number of fragments specified by 5′ and 3′positions or by size in nucleotides, as described above, may beexcluded.

Variants

In other preferred embodiments, variants of OBG3 and gOBG3polynucleotides encoding OBG3 and gOBG3 fragments are envisioned.Variants of polynucleotides, as the term is used herein, arepolynucleotides whose sequence differs from a reference polynucleotide.A variant of a polynucleotide may be a naturally occurring variant suchas a naturally occurring allelic variant, or it may be a variant that isnot known to occur naturally. Such non-naturally occurring variants ofthe polynucleotide may be made by mutagenesis techniques, includingthose applied to polynucleotides, cells or organisms. Generally,differences are limited so that the nucleotide sequences of thereference and the variant are closely similar overall and, in manyregions, identical.

Polynucleotide variants that comprise a sequence substantially differentfrom those described above but that, due to the degeneracy of thegenetic code, still encode OBG3 and gOBG3 polypeptide fragments of thepresent invention are also specifically envisioned. It would also beroutine for one skilled in the art to generate the degenerate variantsdescribed above, for instance, to optimize codon expression for aparticular host (e.g., change codons in the human mRNA to thosepreferred by other mammalian or bacterial host cells).

As stated above, variant polynucleotides may occur naturally, such as anatural allelic variant, or by recombinant methods. By an “allelicvariant” is intended one of several alternate forms of a gene occupyinga given locus on a chromosome of an organism (See, e.g., B. Lewin.(1990) Genes IV, Oxford University Press. New York). Non-naturallyoccurring variants may be produced using art-known muragenesistechniques. Such nucleic acid variants include those produced bynucleotide substitutions, deletions, or additions. The substitutions,deletions, or additions may involve one or more nucleotides. Alterationsin the coding regions may produce conservative or non-conservative aminoacid substitutions, deletions or additions. Especially preferred amongthese are silent substitutions, additions and deletions, which do notalter the properties and activities of an OBG3 or gOBG3 polypeptidefragment of the invention. Also preferred in this regard areconservative substitutions.

Nucleotide changes present in a variant polynucleotide are preferablysilent, which means that they do not alter the amino acids encoded bythe polynucleotide. However, nucleotide changes may also result in aminoacid substitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence.

In cases where the nucleotide substitutions result in one or more aminoacid changes, preferred OBG3 and gOBG3 polypeptide fragments includethose that retain one or more obesity-related activity as described inSection I of the Preferred Embodiments of the Invention.

By “retain the same activities” is meant that the activity measuredusing the polypeptide encoded by the variant OBG3 or gOBG3polynucleotide in assays is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100%, and not more than 101%, 102%, 103%, 104%, 105%, 110%,115%, 120% or 125% of the activity measured using a gOBG3 fragmentdescribed in the Examples Section herein.

By the activity being “increased” is meant that the activity measuredusing the polypeptide encoded by the variant OBG3 or gOBG3polynucleotide in assays is at least 125%, 130%, 135%, 140%, 145%, 150%,155° %, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%,35000, 375%, 400%, 450%, or 500% of the activity measured using a gOBG3fragment described in the Examples Section herein.

By the activity being “decreased” is meant that the activity measuredusing the polypeptide encoded by the variant OBG3 or gOBG3polynucleotide in assays is decreased by at least 25%, 30%, 35%, 40%,45%, or 50% of the activity measured using a gOBG3 fragment described inthe Examples Section herein.

Percent Identity

The present invention is further directed to nucleic acid moleculeshaving sequences at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 93% or99% identical to the polynucleotide sequences of SEQ ID NO:1, SEQ IDNO:3, or SEQ ID NO:5 or fragments thereof that encode a polypeptidehaving obesity-related activity as described in Section I of thePreferred Embodiments of the Invention. Of course, due to the degeneracyof the genetic code, one of ordinary skill in the art will immediatelyrecognize that a large number of the nucleic acid molecules at least50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thenucleic acid sequences shown in SEQ ID NO: 1, SEQ ID NO:3, or SEQ IDNO:5 or fragments thereof will encode a polypeptide having biologicalactivity. In fact, since degenerate variants of these nucleotidesequences all encode the same polypeptide, this will be clear to theskilled artisan even without performing the above described comparisonassay. It will be further recognized in the art that, for such nucleicacid molecules that are not degenerate variants, a reasonable numberwill also encode a polypeptide having biological activity. This isbecause the skilled artisan is fully aware of amino acid substitutionsthat are either less likely or not likely to significantly affectprotein function (e.g., replacing one aliphatic amino acid with a secondaliphatic amino acid), as further described previously in Section I ofthe Preferred Embodiments of the Invention.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the OBG3or gOBG3 fragment. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted, inserted, or substituted with another nucleotide. The querysequence may be an entire sequence or any fragment specified asdescribed herein.

The methods of determining and defining whether any particular nucleicacid molecule or polypeptide is at least 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% identical to a nucleotide sequence of the presentinvention can be done by using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al.,((1990) Comput Appl Biosci 6(3):237-45). In a sequence alignment thequery and subject sequences are both DNA sequences. An RNA sequence canbe compared by first converting U's to T's. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB alignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4. Mismatch Penalty=−1 Joining Penalty=30.Randomization Group Length=0, Cutoff Score=1. Gap Penalty=5, Gap SizePenalty 0.05. Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only nucleotidesoutside the 5′ and 3′ nucleotides of the subject sequence, as displayedby the FASTDB alignment, which are not matched/aligned with the querysequence, are calculated for the purposes of manually adjusting thepercent identity score.

For example, a 90-nucleotide subject sequence is aligned to a100-nucleotide query sequence to determine percent identity. Thedeletions occur at the 5′ end of the subject sequence and therefore, theFASTDB alignment does not show a matched alignment of the first 10nucleotides at 5′ end. The 10 unpaired nucleotides represent 10% of thesequence (number of nucleotides at the 5′ and 3′ ends not matched/totalnumber of nucleotides in the query sequence) so 10% is subtracted fromthe percent identity score calculated by the FASTDB program. If theremaining 90 nucleotides were perfectly matched the final percentidentity would be 90%. In another example, a 90 nucleotide subjectsequence is compared with a 100 nucleotide query sequence. This time thedeletions are internal deletions so that there are no nucleotides on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only nucleotides 5′ and 3′ of thesubject sequence which are not matched/aligned with the query sequenceare manually corrected for. No other manual corrections are made for thepurposes of the present invention.

Fusions

Further included in the present invention are polynucleotides encodingthe polypeptides of the present invention that are fused in frame to thecoding sequences for additional heterologous amino acid sequences. Alsoincluded in the present invention are nucleic acids encodingpolypeptides of the present invention together with additional,non-coding sequences, including for example, but not limited tonon-coding 5′ and 3′ sequences, vector sequence, sequences used forpurification, probing, or priming. For example, heterologous sequencesinclude transcribed, nontranslated sequences that may play a role intranscription, and mRNA processing, for example, ribosome binding andstability of mRNA. The heterologous sequences may alternatively compriseadditional coding sequences that provide additional functionalities.Thus, a nucleotide sequence encoding a polypeptide may be fused to a tagsequence, such as a sequence encoding a peptide that facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the tag amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. For instance,hexa-histidine provides for convenient purification of the fusionprotein (See, Gentz et al., (1989) Proc Natl Acad Sci USA 86(3):821-4).The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein (See, Wilson et al., (1984) Cell 37(3):767-78). As discussedabove, other such fusion proteins include OBG3 or gOBG3 fragment cDNAfused to Fc at the N- or C-terminus.

III. Recombinant Vectors of the Invention

The term “vector” is used herein to designate either a circular or alinear DNA or RNA molecule, that is either double-stranded orsingle-stranded, and that comprises at least one polynucleotide ofinterest that is sought to be transferred in a cell host or in aunicellular or multicellular host organism.

The present invention relates to recombinant vectors comprising any oneof the polynucleotides described herein.

The present invention encompasses a family of recombinant vectors thatcomprise polynucleotides encoding OBG3 polypeptide fragments of theinvention.

In a first preferred embodiment, a recombinant vector of the inventionis used to amplify the inserted polynucleotide in a suitable cell host,this polynucleotide being amplified every time that the recombinantvector replicates. The inserted polynucleotide can be one that encodesgOBG3 polypeptide fragments of the invention.

A second preferred embodiment of the recombinant vectors according tothe invention consists of expression vectors comprising polynucleotidesencoding OBG3 polypeptide fragments of the invention. Within certainembodiments, expression vectors are employed to express an OBG3 fragmentof the invention, preferably a modified OBG3 fragment described in thepresent invention, which can be then purified and, for example, be usedas a treatment for obesity-related diseases, or simply to reduce bodymass of individuals.

Expression requires that appropriate signals are provided in thevectors, said signals including various regulatory elements, such asenhancers/promoters from both viral and mammalian sources, that driveexpression of the genes of interest in host cells. Dominant drugselection markers for establishing permanent, stable, cell clonesexpressing the products are generally included in the expression vectorsof the invention, as they are elements that link expression of the drugselection markers to expression of the polypeptide.

More particularly, the present invention relates to expression vectorswhich include nucleic acids encoding an OBG3 fragment of the invention,or a modified OBG3 fragment as described herein, or variants orfragments thereof, under the control of a regulatory sequence selectedamong OBG3 polypeptide fragments, or alternatively under the control ofan exogenous regulatory sequence.

Consequently, preferred expression vectors of the invention are selectedfrom the group consisting of: (a) an OBG3 fragment regulatory sequenceand driving the expression of a coding polynucleotide operably linkedthereto; and (b) an OBG3 fragment coding sequence of the invention,operably linked to regulatory sequences allowing its expression in asuitable cell host and/or host organism.

Some of the elements that can be found in the vectors of the presentinvention are described in further detail in the following sections.

1) General Features of the Expression Vectors of the Invention:

A recombinant vector according to the invention comprises, but is notlimited to, a YAC (Yeast Artificial Chromosome), a BAC (BacterialArtificial Chromosome), a phage, a phagemid, a cosmid, a plasmid, oreven a linear DNA molecule which may consist of a chromosomal,non-chromosomal, semi-synthetic or synthetic DNA. Such a recombinantvector can comprise a transcriptional unit comprising an assembly of:

(1) a genetic element or elements having a regulatory role in geneexpression, for example promoters or enhancers. Enhancers are cis-actingelements of DNA, usually from about to 300 bp in length that act on thepromoter to increase the transcription:

(2) a structural or coding sequence which is transcribed into mRNA andeventually translated into a polypeptide, said structural or codingsequence being operably linked to the regulatory elements described in(1): and

(3) appropriate transcription initiation and termination sequences.Structural units intended for use in yeast or eukaryotic expressionsystems preferably include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, when arecombinant protein is expressed without a leader or transport sequence,it may include a N-terminal residue. This residue may or may not besubsequently cleaved from the expressed recombinant protein to provide afinal product.

Generally, recombinant expression vectors will include origins ofreplication, selectable markers permitting transformation of the hostcell, and a promoter derived from a highly expressed gene to directtranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences, and preferably a leader sequencecapable of directing secretion of the translated protein into theperiplasmic space or the extracellular medium. In a specific embodimentwherein the vector is adapted for transfecting and expressing desiredsequences in mammalian host cells, preferred vectors will comprise anorigin of replication in the desired host, a suitable promoter andenhancer, and also any necessary ribosome binding sites, polyadenylationsites, splice donor and acceptor sites, transcriptional terminationsequences, and 5′-flanking non-transcribed sequences. DNA sequencesderived from the SV40 viral genome, for example SV40 origin, earlypromoter, enhancer, splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

2) Regulatory Elements

Promoters

The suitable promoter regions used in the expression vectors of thepresent invention are chosen taking into account the cell host in whichthe heterologous gene is expressed. The particular promoter employed tocontrol the expression of a nucleic acid sequence of interest is notbelieved to be important, so long as it is capable of directing theexpression of the nucleic acid in the targeted cell. Thus, where a humancell is targeted, it is preferable to position the nucleic acid codingregion adjacent to and under the control of a promoter that is capableof being expressed in a human cell, such as, for example, a human or aviral promoter. The promoter used may be constitutive or inducible.

A suitable promoter may be heterologous with respect to the nucleic acidfor which it controls the expression or alternatively can be endogenousto the native pol-nucleotide containing the coding sequence to beexpressed. Additionally, the promoter is generally heterologous withrespect to the recombinant vector sequences within which the constructpromoter/coding sequence has been inserted.

Promoter regions can be selected from any desired gene using, forexample, CAT (chloramphenicol transferase) vectors and more preferablypKK232-8 and pCM7 vectors. Preferred bacterial promoters are the LacI,LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt,lambda PR. PL and trp promoters (EP 0036776), the polyhedrin promoter,or the p10 protein promoter from baculovirus (Kit Novagen) (Smith etal., (19S3) Mol Cell Biol 3(12):2156-65: O'Reilly et al. 1992), thelambda PR promoter or also the trc promoter.

Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40. LTRs from retrovirus, and mouse metallothionein-L.In addition, promoters specific for a particular cell type may bechosen, such as those facilitating expression in adipose tissue, muscletissue, or liver. Selection of a convenient vector and promoter is wellwithin the level of ordinary skill in the art.

The choice of a promoter is well within the ability of a person skilledin the field of genetic engineering. For example, one may refer toSambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), or also to theprocedures described by Fuller et al. (1996) Immunology in CurrentProtocols in Molecular Biology.

Other Regulatory Elements

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

Vectors containing the appropriate DNA sequence as described above canbe utilized to transform an appropriate host to allow the expression ofthe desired polypeptide or polynucleotide.

3) Selectable Markers

Such markers would confer an identifiable change to the cell permittingeasy identification of cells containing the expression construct. Theselectable marker genes for selection of transformed host cells arepreferably dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, TRP1 for S. cerevisiae or tetracycline, rifampicin orampicillin resistance in E. coli, or leven saccharase for mycobacteria,this latter marker being a negative selection marker.

4) Preferred Vectors

Bacterial Vectors

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and a bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of pBR322 (ATCC 37017). Such commercialvectors include, but are not limited to pKK223-3 (Pharmacia. Uppsala,Sweden) and pGEM1 (Promega Biotec, Madison. Wis., USA).

Large numbers of other suitable vectors are known to those of skill inthe art, and are commercially available, such as the following bacterialvectors: pTrc-His, pET30-His, pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10,phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A,pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia): pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3,pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).

Baculovirus Vectors

A suitable vector for the expression of polypeptides of the invention isa baculovirus vector that can be propagated in insect cells and ininsect cell lines. A specific suitable host vector system is thepVL1392/1393 baculovirus transfer vector (Pharmingen) that is used totransfect the SF9 cell line (ATCC No CRL 1711) which is derived fromSpodoptera frugiperda. Further suitable baculovirus vectors are known tothose skilled in the art, for example, FastBacHT. Other suitable vectorsfor the expression of an APM1 globular head polypeptide in a baculovirusexpression system include, but are not limited to, those described byChai et al. (1993; Biotechnol Appl Biochem. December: 18 (Pt 3):259-73);Vlasak et al. (1983; Eur J Biochem September 1; 135(1):123-6); andLenhard et al. (1996; Gene Mar 9; 169(2):117-90).

Mammalian Vectors

Further suitable vectors for the expression of polypeptides of theinvention are mammalian vectors. A number of suitable vector systems areknown to those skilled in the art, for example, pcDNA4HisMax,pcDNA3.1Hygro-His and pcDNA3.1Hygro.

Viral Vectors

In one specific embodiment, the vector is derived from an adenovirus.Preferred adenovirus vectors according to the invention are thosedescribed by Feldman and Steg (1996; Semin Interv Cardiol 1(3):203-8) orOhno et al. (1994; Science 265(5173):781-4). Another preferredrecombinant adenovirus according to this specific embodiment of thepresent invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) oran adenovirus of animal origin (French patent application No.FR-93.05954).

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery systems of choice for thetransfer of exogenous polynucleotides in vivo, particularly to mammals,including humans. These vectors provide efficient delivery of genes intocells, and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host.

Particularly preferred retroviruses for the preparation or constructionof retroviral in vitro or in vivo gene delivery vehicles of the presentinvention include retroviruses selected from the group consisting ofMink-Cell Focus Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus and Rous Sarcoma virus. Particularlypreferred Murine Leukemia Viruses include the 4070A and the 1504Aviruses. Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCCNo VR-590), Rauscher (ATCC No VR-998) and Molonev Murine Leukemia Virus(ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferredRous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657,VR-726, VR-659 and VR-728). Other preferred retroviral vectors are thosedescribed in Roth et al. (1996), PCT Application No WO 93/25234, PCTApplication No WO 94/06920, Roux et al., ((1989) Proc Natl Acad Sci USA86(23):9079-83), Julan et al., (1992) J. Gen. Virol. 3:3251-3255 andNeda et al. ((1991) J Biol Chem 266(22): 14143-6).

Yet another viral vector system that is contemplated by the inventionconsists of the adeno-associated virus (AAV). The adeno-associated virusis a naturally occurring defective virus that requires another virus,such as an adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle (Muzyczka et al., (1992) CurrTop Microbiol Immunol 158:97-129). It is also one of the few virusesthat may integrate its DNA into non-dividing cells, and exhibits a highfrequency of stable integration (Flotte et al., (1992) Am J Respir CellMol Biol 7(3):349-56; Samulski et al., (1989) J Virol 63(9):3822-8:McLaughlin et al., (1989) Am J Hum Genet. 59:561-569). One advantageousfeature of AAV derives from its reduced efficacy for transducing primarycells relative to transformed cells.

5) Delivery of the Recombinant Vectors

In order to effect expression of the polynucleotides of the invention,these constructs must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transforming celllines, or in vivo or ex vivo, as in the treatment of certain diseasestates.

One mechanism is viral infection where the expression construct isencapsulated in an infectious viral particle.

Several non-viral methods for the transfer of polynucleotides intocultured mammalian cells are also contemplated by the present invention,and include, without being limited to, calcium phosphate precipitation(Graham et al., (1973) Virology 54(2):536-9: Chen et al., (1987) MolCell Biol 7(8):2745-52), DEAE-dextran (Gopal, (1985) Mol Cell Biol5(5):1188-90), electroporation (Tur-Kaspa et al. (1986) Mol Cell Biol6(2):716-S: Potter et al., (1984) Proc Natl Acad Sci USA81(22):7161-5.), direct microinjection (Harland et al. (1985) J CellBiol 101(3):1094-9), DNA-loaded liposomes (Nicolau et al., (1982)Biochim Biophys Acta 721(2):185-90; Fraley et al., (1979) Proc Natl AcadSci USA 76(7):3348-52), and receptor-mediated transfection (Wu and Wu,(1987) J Biol Chem 262(10):4429-32: Wu and Wu (1988) Biochemistry27(3):887-92). Some of these techniques may be successfully adapted forin vivo or ex vivo use.

Once the expression polynucleotide has been delivered into the cell, itmay be stably integrated into the genome of the recipient cell. Thisintegration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle.

One specific embodiment for a method for delivering a protein or peptideto the interior of a cell of a vertebrate in vivo comprises the step ofintroducing a preparation comprising a physiologically acceptablecarrier and a naked polynucleotide operatively coding for thepolypeptide of interest into the interstitial space of a tissuecomprising the cell, whereby the naked polynucleotide is taken up intothe interior of the cell and has a physiological effect. This isparticularly applicable for transfer in vitro but it may be applied toin vivo as well.

Compositions for use in vitro and in vivo comprising a “naked”polynucleotide are described in PCT application No. WO 90/11092 (VicalInc.) and also in PCT application No. WO 95/11307 (Institut Pasteur,INSERM. Universite d'Ottawa) as well as in the articles of Tascon et al.(1996) Nature Medicine 2(8):888-892 and of Huygen et al. ((1996) Nat Med2(8):893-8).

In still another embodiment of the invention, the transfer of a nakedpolynucleotide of the invention, including a polynucleotide construct ofthe invention, into cells may be proceeded with a particle bombardment(biolistic), said particles being DNA-coated microprojectilesaccelerated to a high velocity allowing them to pierce cell membranesand enter cells without killing them, such as described by Klein et al.((1990) Curr Genet February:17(2):97-103).

In a further embodiment, the polynucleotide of the invention may beentrapped in a liposome (Ghosh and Bacchawat. (1991) Targeted Diagn Ther4:87-103: Wong et al., (1980) Gene 10:87-94: Nicolau et al. (1987)Methods Enzymol 149:157-76). These liposomes may further be targeted tocells expressing LSR by incorporating leptin, triglycerides. ACRP30, orother known LSR ligands into the liposome membrane.

In a specific embodiment, the invention provides a composition for thein vivo production of an APM1 globular head polypeptide describedherein. It comprises a naked polynucleotide operatively coding for thispolypeptide, in solution in a physiologically acceptable carrier, andsuitable for introduction into a tissue to cause cells of the tissue toexpress the said polypeptide.

The amount of vector to be injected to the desired host organism variesaccording to the site of injection. As an indicative dose, it will beinjected between 0.1 and 100 μg of the vector in an animal body,preferably a mammal body, for example a mouse body.

In another embodiment of the vector according to the invention, it maybe introduced in vitro in a host cell, preferably in a host cellpreviously harvested from the animal to be treated and more preferably asomatic cell such as a muscle cell. In a subsequent step, the cell thathas been transformed with the vector coding for the desired APM1globular head polypeptide or the desired fragment thereof isreintroduced into the animal body in order to deliver the recombinantprotein within the body either locally or systemically.

IV. Recombinant Cells of the Invention

Another object of the invention consists of host cells recombinant for,i.e., that have been transformed or transfected with one of thepolynucleotides described herein, and more precisely a polynucleotidecomprising a polynucleotide encoding an OBG3 polypeptide fragment of theinvention such as any one of those described in “Polynucleotides of theInvention”. These polynucleotides can be present in cells as a result oftransient or stable transfection. The invention includes host cells thatare transformed (prokaryotic cells) or that are transfected (eukaryoticcells) with a recombinant vector such as any one of those described in“Recombinant Vectors of the Invention”.

Generally, a recombinant host cell of the invention comprises at leastone of the polynucleotides or the recombinant vectors of the inventionthat are described herein.

Preferred host cells used as recipients for the recombinant vectors ofthe invention are the following

a) Prokaryotic host cells: Escherichia coli strains (I.E. DH5-ctstrain), Bacillus subtilis, Salmonella typhimurium, and strains fromspecies like Pseudomonas, Streptomyces and Staphlococcus, and

b) Eukaryotic host cells: HeLa cells (ATCC No CCL2: No CCL2.1: NCCL2.2),Cv 1 cells (ATCC No CCL70). COS cells (ATCC No CRL 1650: No CRL 1651).Sf-9 cells (ATCC No CRL 1711). C127 cells (ATCC No CRL-1804). 3T3 (ATCCNo CRL-6361). CHO (ATCC No CCL-61), human kidney 293 (ATCC No 45504: NoCRL-1573). BHK (ECACC No 84100501; NO 84111301). PLC cells. HepG2, andHep3B.

The constructs in the host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth of the host to anappropriate cell density, the selected promoter is induced byappropriate means, such as temperature shift or chemical induction, andcells are cultivated for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in the expression of proteins can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents. Such methods arewell known by the skilled artisan.

Further, according to the invention, these recombinant cells can becreated in vitro or in vivo in an animal, preferably a mammal, mostpreferably selected from the group consisting of mice, rats, dogs, pigs,sheep, cattle, and primates, not to include humans. Recombinant cellscreated in vitro can also be later surgically implanted in an animal,for example. Methods to create recombinant cells in vivo in animals arewell known in the art.

The present invention also encompasses primary, secondary, andimmortalized homologously recombinant host cells of vertebrate origin,preferably mammalian origin and particularly human origin, that havebeen engineered to: a) insert exogenous (heterologous) polynucleotidesinto the endogenous chromosomal DNA of a targeted gene, b) deleteendogenous chromosomal DNA, and, or c) replace endogenous chromosomalDNA with exogenous polynucleotides. Insertions, deletions, and/orreplacements of polynucleotide sequences may be to the coding sequencesof the targeted gene and/or to regulatory regions, such as promoter andenhancer sequences, operably associated with the targeted gene.

The present invention further relates to a method of making ahomologously recombinant host cell in vitro or in vivo, wherein theexpression of a targeted gene not normally expressed in the cell isaltered. Preferably the alteration causes expression of the targetedgene under normal growth conditions or under conditions suitable forproducing the polypeptide encoded by the targeted gene. The methodcomprises the steps of: (a) transfecting the cell in vitro or in vivowith a polynucleotide construct, the polynucleotide constructcomprising; (i) a targeting sequence; (ii) a regulatory sequence and/ora coding sequence: and (iii) an unpaired splice donor site, ifnecessary, thereby producing a transfected cell: and (b) maintaining thetransfected cell in vitro or in vivo under conditions appropriate forhomologous recombination.

The present invention further relates to a method of altering theexpression of a targeted gene in a cell in vitro or in vivo wherein thegene is not normally expressed in the cell, comprising the steps of: (a)transfecting the cell in vitro or in vivo with a polynucleotideconstruct, the polynucleotide construct comprising: (i) a targetingsequence: (ii) a regulatory sequence and/or a coding sequence: and (iii)an unpaired splice donor site, if necessary, thereby producing atransfected cell: and (b) maintaining the transfected cell in vitro orin vivo under conditions appropriate for homologous recombination,thereby producing a homologously recombinant cell; and (c) maintainingthe homologously recombinant cell in vitro or in vivo under conditionsappropriate for expression of the gene.

The present invention further relates to a method of making apolypeptide of the present invention by altering the expression of atargeted endogenous gene in a cell in vitro or in vivo wherein the geneis not normally expressed in the cell, comprising the steps of: a)transfecting the cell in vitro with a polynucleotide construct, thepolynucleotide construct comprising: (i) a targeting sequence; (ii) aregulatory sequence and/or a coding sequence; and (iii) an unpairedsplice donor site, if necessary, thereby producing a transfected cell;(b) maintaining the transfected cell in vitro or in vivo underconditions appropriate for homologous recombination, thereby producing ahomologously recombinant cell; and c) maintaining the homologouslyrecombinant cell in vitro or in vivo under conditions appropriate forexpression of the gene thereby making the polypeptide.

The present invention further relates to a polynucleotide construct thatalters the expression of a targeted gene in a cell type in which thegene is not normally expressed. This occurs when a polynucleotideconstruct is inserted into the chromosomal DNA of the target cell,wherein the polynucleotide construct comprises: a) a targeting sequence:b) a regulatory sequence and/or coding sequence: and c) an unpairedsplice-donor site, if necessary. Further included are polynucleotideconstructs, as described above, wherein the construct further comprisesa polynucleotide that encodes a polypeptide and is in-frame with thetargeted endogenous gene after homologous recombination with chromosomalDNA.

The compositions may be produced, and methods performed, by techniquesknown in the art, such as those described in U.S. Pat. Nos. 6,054,288;6,048, 729; 6,048,724; 6,048, 524; 5,994, 127; 5,968, 502; 5,965,125;5,869,239; 5,817,789; 5,783,385; 5,733,761; 5,641,670; 5,580,734;International Publication Nos; WO96/29411, WO 94/12650; and scientificarticles described by Koller et al., (1994) Annu. Rev. Immunol.10:705-730: the disclosures of each of which are incorporated byreference in their entireties).

The OBG3 gene expression in mammalian, and typically human, cells may berendered defective, or alternatively it may be enhanced, with theinsertion of an OBG3 genomic or cDNA sequence with the replacement ofthe OBG3 gene counterpart in the genome of an animal cell by an OBG3polynucleotide according to the invention. These genetic alterations maybe generated by homologous recombination events using specific DNAconstructs that have been previously described.

One kind of host cell that may be used are mammalian zygotes, such asmurine zygotes. For example, murine zygotes may undergo microinjectionwith a purified DNA molecule of interest, for example a purified DNAmolecule that has previously been adjusted to a concentration range from1 ng/ml—for BAC inserts—3 ng/μl—for P1 bacteriophage inserts—in 10 mMTris-HCl. pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine,and 70 μM spermidine. When the DNA to be microinjected has a large size,polyamines and high salt concentrations can be used in order to avoidmechanical breakage of this DNA, as described by Schedl et al ((1993)Nature 362(6417):258-61).

Any one of the polynucleotides of the invention, including the DNAconstructs described herein, may be introduced in an embryonic stem (ES)cell line, preferably a mouse ES cell line. ES cell lines are derivedfrom pluripotent, uncommitted cells of the inner cell mass ofpre-implantation blastocysts. Preferred ES cell lines are the following:ES-E14TG2a (ATCC No. CRL-1821), ES-D3 (ATCC No. CRL1934 and No.CRL-11632), YS001 (ATCC No. CRL-1776). 36.5 (ATCC No. CRL-11116). Tomaintain ES cells in an uncommitted state, they are cultured in thepresence of growth inhibited feeder cells that provide the appropriatesignals to preserve this embryonic phenotype and serve as a matrix forES cell adherence. Preferred feeder cells are primary embryonicfibroblasts that are established from tissue of day 13-day 14 embryos ofvirtually any mouse strain, that are maintained in culture, such asdescribed by Abbondanzo et al. (1993; Methods Enzymol 225:803-23) andare inhibited in growth by irradiation, such as described by Robertson((1987) Embryo-derived stern cell lines. In: E. J. Robertson Ed.Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, IRLPress. Oxford), or by the presence of an inhibitory concentration of L,such as described by Pease and Williams (1990; Exp Cell Res190(2):209-11).

The constructs in the host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth of the host to anappropriate cell density, the selected promoter is induced byappropriate means, such as temperature shift or chemical induction, andcells are cultivated for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in the expression of proteins can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents. Such methods arewell known by the skilled artisan.

IV. Transgenic Animals

The present invention also provides methods and compositions for thegeneration of non-human animals and plants that express recombinant OBG3polypeptides, i.e., recombinant OBG3 fragments or full-length OBG3polypeptides. The animals or plants can be transgenic, i.e., each oftheir cells contains a gene encoding the OBG3 polypeptide, or,alternatively, a polynucleotide encoding the polypeptide can beintroduced into somatic cells of the animal or plant, e.g., into mammarysecretory epithelial cells of a mammal. In preferred embodiments, thenon-human animal is a mammal such as a cow, sheep, goat, pig, or rabbit.

Methods of making transgenic animals such as mammals are well mown tothose of skill in the art, and any such method can be used in thepresent invention. Briefly, transgenic mammals can be produced, e.g., bytransfecting a pluripotential stem cell such as an ES cell with apolynucleotide encoding a polypeptide of interest. Successfullytransformed ES cells can then be introduced into an early stage embryothat is then implanted into the uterus of a mammal of the same species.In certain cases, the transformed (“transgenic”) cells will comprisepart of the germ line of the resulting animal, and adult animalscomprising the transgenic cells in the germ line can then be mated toother animals, thereby eventually producing a population of transgenicanimals that have the transgene in each of their cells, and which canstably transmit the transgene to each of their offspring. Other methodsof introducing the polynucleotide can be used, for example introducingthe polynucleotide encoding the polypeptide of interest into afertilized egg or early stage embryo via microinjection. Alternatively,the transgene may be introduced into an animal by infection of zygoteswith a retrovirus containing the transgene (Jaenisch, R. (1976) ProcNatl Acad Sci USA 73, 1260-1264). Methods of making transgenic mammalsare described, e.g., in Wall et al. (1992) J Cell Biochem 1992 49(2):113-20; Hogan, et al. (1986) in Manipulating the Mouse Embryo: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; in WO 91/08216; or in U.S. Pat. No. 4,736,866.

In a preferred method, the polynucleotides are microinjected into thefertilized oocyte. Typically, fertilized oocytes are microinjected usingstandard techniques, and then cultured in vitro until a“pre-implantation embryo” is obtained. Such pre-implantation embryospreferably contain approximately 16 to 150 cells. Methods for culturingfertilized oocytes to the pre-implantation stage are described, e.g., byGordon et al. ((1984) Methods in Enzymology, 101, 414); Hogan et al.((1986) in Manipulating the Mouse Embryo: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) (for the mouseembryo); Hammer et al. ((1985) Nature, 315, 680) (for rabbit and porcineembryos); Gandolfi et al. ((1987) J. Reprod. Fert. 81, 23-28); Rexroadet al. ((1988) J. Anim. Sci. 66, 947-953) (for ovine embryos); andEyestone et al. ((1989) J. Reprod. Fert. 85, 715-720: Camous et al.((1984) J. Reprod. Fert. 72, 779-785); and Heyman et al. ((1987)Theriogenology 27, 5968) (for bovine embryos); the disclosures of eachof which are incorporated herein in their entireties. Pre-implantationembryos are then transferred to an appropriate female by standardmethods to permit the birth of a transgenic or chimeric animal dependingupon the stage of development when the transgene is introduced.

As the frequency of transgene incorporation is often low, the detectionof transgene integration in pre-implantation embryos is often desirableusing any of the herein-described methods. Any of a number of methodscan be used to detect the presence of a transgene in a pre-implantationembryo. For example, one or more cells may be removed from thepre-implantation embryo, and the presence or absence of the transgene inthe removed cell or cells can be detected using any standard method e.g.PCR. Alternatively, the presence of a transgene can be detected in uteroor post partum using standard methods.

In a particularly preferred embodiment of the present invention,transgenic mammals are generated that secrete recombinant OBG3polypeptides in their milk. As the mammary gland is a highly efficientprotein-producing organ, such methods can be used to produce proteinconcentrations in the gram per liter range, and often significantlymore. Preferably, expression in the mammary gland is accomplished byoperably linking the polynucleotide encoding the OBG3 polypeptide to amammary gland specific promoter and, optionally, other regulatoryelements. Suitable promoters and other elements include, but are notlimited to, those derived from mammalian short and long WAP, alpha,beta, and kappa, casein, alpha and beta lactoglobulin, beta-CN 5′ genes,as well as the mouse mammary tumor virus (MMTV) promoter. Such promotersand other elements may be derived from any mammal, including, but notlimited to, cows, goats, sheep, pigs, mice, rabbits, and guinea pigs.Promoter and other regulatory sequences, vectors, and other relevantteachings are provided, e.g., by Clark (1998) J Mammary Gland BiolNeopiasia 3:337-50; Jost et al. (1999) Nat Biotechnol 17:160-4; U.S.Pat. Nos. 5,994,616; 6,140,552; 6.013, 857; Sohn et al. (1999) DNA CellBiol. 18:845-52: Kim et al. (1999) Biochem (Japan) 126:320-5; Soulier etal. (1999) Euro J Biochem 260:533-9: Zhang et al. (1997) Chin J Biotech13:271-6: Rijnkels et al. (1998) Transgen Res 7:5-14: Korhonen et al.(1997) Euro J Biochem 245:482-9: Uusi-Oukari et al. (1997) Transgen Res6:75-84: Hitchin et al. (1996) Prot Expr Purif 7:247-52 Platenburg etal. (1994) Transgen Res 3:99-108: Heng-Cherl et al. (1993) AnimalBiotech 4:89-107: and Christa et al. (2000) Euro J Biochem 267:1665-71;the entire disclosures of each of which is herein incorporated byreference.

In another embodiment, the polypeptides of the invention can be producedin milk by introducing polynucleotides encoding the polypeptides intosomatic cells of the mammary gland in vivo, e.g. mammary secretingepithelial cells. For example, plasmid DNA can be infused through thenipple canal, e.g., in association with DEAE-dextran (see, e.g., Hens etal. (2000)

Biochim. Biophys. Acta 1523:161-171), in association with a ligand thatcan lead to receptor-mediated endocytosis of the construct (see. e.g.Sobolev et al. (1998) 273:7928-33) or in a viral vector such as aretroviral vector, e.g., the Gibbon ape leukemia virus (see, e.g. Archeret al. (1994) PNAS 91:6840-6844). In any of these embodiments, thepolynucleotide may be operably linked to a mammary gland specificpromoter, as described above, or, alternatively, any strongly expressingpromoter such as CMV or MoMLV LTR.

The suitability of any vector, promoter, regulatory element, etc, foruse in the present invention can be assessed beforehand by transfectingcells such as mammary epithelial cells, e.g. MacT cells (bovine mammaryepithelial cells) or GME cells (gcat mammary epithelial cells), in vitroand assessing the efficiency of transfection and expression of thetransgene in the cells.

For in vivo administration, the polynucleotides can be administered inany suitable formulation, at any of a range of concentrations (e.g.1-500 μg/ml, preferably 50-100 μg/ml), at any volume (e.g. 1-100 ml,preferably 1 to 20 ml), and can be administered any number of times(e.g. 1, 2, 3, 5, or 10 times), at any frequency (e.g., every 1, 2, 3,5, 10, or any number of days). Suitable concentrations, frequencies,modes of administration, etc, will depend upon the particularpolynucleotide, vector, animal, etc., and can readily be determined byone of skill in the art.

In a preferred embodiment, a retroviral vector such as Gibbon apeleukemia viral vector is used, as described in Archer et al. ((1994)PNAS 91:6840-6844). As retroviral infection typically requires celldivision, cell division in the mammary glands can be stimulated inconjunction with the administration of the vector, e.g., using a factorsuch as estrodiol benzoate, progesterone, reserpine, or dexamethasone.Further, retroviral and other methods of infection can be facilitatedusing accessory compounds such as polybrene.

In any of the herein-described methods for obtaining OBG3 polypeptidesfrom milk, the quantity of milk obtained, and thus the quantity of OBG3polypeptides produced, can be enhanced using any standard method oflactation induction, e.g., using hexestrol, estrogen, and/orprogesterone.

The polynucleotides used in such embodiments can either encode afull-length OBG3 polypeptide or an OBG3 fragment. Typically, the encodedpolypeptide will include a signal sequence to ensure the secretion ofthe protein into the milk. Where a full-length OBG3 sequence is used,the full-length protein can, e.g., be isolated from milk and cleaved invitro using a suitable protease. Alternatively, a secondprotease-encoding polynucleotide can be introduced into the animal orinto the mammary gland cells, whereby expression of the protease resultsin the cleavage of the OBG3 polypeptide in vivo, thereby allowing thedirect isolation of OBG3 fragments from milk.

V. Pharmaceutical or Physiologically Acceptable Compositions of theInvention

The OBG3 and gOBG3 polypeptide fragments of the invention can beadministered to non-human animals and/or humans, alone or inpharmaceutical or physiologically acceptable compositions where they aremixed with suitable carriers or excipient(s). The pharmaceutical orphysiologically acceptable composition is then provided at atherapeutically effective dose. A therapeutically effective dose refersto that amount of OBG3 or gOBG3 fragment sufficient to result inprevention or amelioration of symptoms or physiological status ofobesity-related diseases or disorders as determined by the methodsdescribed herein. A therapeutically effective dose can also refer to theamount of OBG3 or gOBG3 fragment necessary for a reduction in weight ora prevention of an increase in weight or prevention of an increase inthe rate of weight gain in persons desiring this affect for cosmeticreasons. A therapeutically effective dosage of an OBG3 or gOBG3 fragmentof the invention is that dosage that is adequate to promote weight lossor weight gain with continued periodic use or administration. Techniquesfor formulation and administration of OBG3 polypeptide fragments may befound in “Remington's Pharmaceutical Sciences,” Mack Publishing Co.Easton, Pa., latest edition.

Other diseases or disorders that OBG3 polypeptide fragments of theinvention could be used to treat or prevent include, but are not limitedto, obesity and obesity-related diseases and disorders such as obesity,impaired glucose tolerance, insulin resistance, atherosclerosis,atheromatous disease, heart disease, hypertension, stroke, Syndrome X,Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) andInsulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).Diabetes-related complications to be treated by the methods of theinvention include microangiopathic lesions, ocular lesions, retinopathy,neuropathy, and renal lesions. Heart disease includes, but is notlimited to, cardiac insufficiency, coronary insufficiency, and highblood pressure. Other obesity-related disorders to be treated bycompounds of the invention include hyperlipidemia and hyperuricemia. Yetother obesity-related diseases or disorders of the invention includecachexia, wasting, AIDS-related weight loss, cancer-related weight loss,anorexia, and bulimia. The OBG3 or gOBG3 polypeptide fragments may alsobe used to enhance physical performance during work or exercise orenhance a feeling of general well-being. Physical performance activitiesinclude walking, running, jumping, lifting and/or climbing.

The OBG3 or gOBG3 polypeptide fragments or antagonists thereof may alsobe used to treat dyslexia, attention-deficit disorder (ADD),attention-deficit hyperactivity disorder (ADHD), and psychiatricdisorders such as schizophrenia by modulating fatty acid metabolism,more specifically, the production of certain long-chain polyunsaturatedfatty acids.

It is expressly considered that the OBG3 or gOBG3 polypeptide fragmentsof the invention may be provided alone or in combination with otherpharmaceutically or physiologically acceptable compounds. Othercompounds useful for the treatment of obesity and other diseases anddisorders are currently well-known in the art.

In a preferred embodiment, the OBG3 or gOBG3 polypeptide fragments areuseful for, and used in, the treatment of insulin resistance anddiabetes using methods described herein and known in the art. Moreparticularly, a preferred embodiments relates to process for thetherapeutic modification and regulation of glucose metabolism in ananimal or human subject, which comprises administering to a subject inneed of treatment (alternatively on a timed daily basis) an OBG or OBG3polypeptide fragment (or polynucleotide encoding said polypeptide) indosage amount and for a period sufficient to reduce plasma glucoselevels in said animal or human subject.

Further preferred embodiments relate to methods for the prophylaxis ortreatment of diabetes comprising administering to a subject in need oftreatment (alternatively on a timed daily basis) an OBG or OBG3polypeptide fragment (or polynucleotide encoding said polypeptide) indosage amount and for a period sufficient to reduce plasma glucoselevels in said animal or human subject.

Routes of Administration.

Suitable routes of administration include oral, nasal, rectal,transmucosal, or intestinal administration, parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocularinjections using methods known in the art. A particularly useful methodof administering compounds for promoting weight loss involves surgicalimplantation, for example into the abdominal cavity of the recipient, ofa device for delivering OBG3 or gOBG3 polypeptide fragments over anextended period of time. Other particularly preferred routes ofadministration are aerosol and depot formulation. Sustained releaseformulations, particularly depot, of the invented medicaments areexpressly contemplated.

Composition/Formulation

Pharmaceutical or physiologically acceptable compositions andmedicaments for use in accordance with the present invention may beformulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries. Properformulation is dependent upon the route of administration chosen.

Certain of the medicaments described herein will include apharmaceutically or physiologically acceptable carrier and at least onepolypeptide that is a OBG3 polypeptide fragment of the invention. Forinjection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer suchas a phosphate or bicarbonate buffer. For transmucosal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

Pharmaceutical or physiologically acceptable preparations that can betaken orally include push-fit capsules made of gelatin, as well as soft,sealed capsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredients inadmixture with fillers such as lactose, binders such as starches, and/orlubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added. Allformulations for oral administration should be in dosages suitable forsuch administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable gaseous propellant, e.g., carbon dioxide. In the case of apressurized aerosol the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin, for use in an inhaler or insufflator, may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical or physiologically acceptable formulations for parenteraladministration include aqueous solutions of the active compounds inwater-soluble form. Aqueous suspensions may contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents that increase the solubility ofthe compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder or lyophilizedform for constitution with a suitable vehicle, such as sterilepyrogen-free water, before use.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days.

Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for protein stabilization maybe employed.

The pharmaceutical or physiologically acceptable compositions also maycomprise suitable solid or gel phase carriers or excipients. Examples ofsuch carriers or excipients include but are not limited to calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, and polymers such as polyethylene glycols.

Effective Dosage.

Pharmaceutical or physiologically acceptable compositions suitable foruse in the present invention include compositions wherein the activeingredients are contained in an effective amount to achieve theirintended purpose. More specifically, a therapeutically effective amountmeans an amount effective to prevent development of or to alleviate theexisting symptoms of the subject being treated. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes orencompasses a concentration point or range shown to increase leptin orlipoprotein uptake or binding in an in vitro system. Such informationcan be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms in a patient. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50, (the dose lethal to 50% of the testpopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio between LD5Oand ED5O. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from these cell culture assays and animal studies canbe used in formulating a range of dosage for use in humans. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED50, with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active compound which are sufficient to maintain orprevent weight loss or gain, depending on the particular situation.Dosages necessary to achieve these effects will depend on individualcharacteristics and route of administration.

Dosage intervals can also be determined using the value for the minimumeffective concentration. Compounds should be administered using aregimen that maintains plasma levels above the minimum effectiveconcentration for 10-90% of the time, preferably between 30-90%; andmost preferably between 50-90%. In cases of local administration orselective uptake, the effective local concentration of the drug may notbe related to plasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

A preferred dosage range for the amount of an OBG3 polypeptide fragmentof the invention, which can be administered on a daily or regular basisto achieve desired results, including a reduction in levels ofcirculating plasma triglyceride-rich lipoproteins, range from 0.01-0.5mg/kg body mass. A more preferred dosage range is from 0.05-0.1 mg/kg.Of course, these daily dosages can be delivered or administered in smallamounts periodically during the course of a day. It is noted that thesedosage ranges are only preferred ranges and are not meant to be limitingto the invention.

VI. Methods of Treatment

Treatment of mice with gOBG3 polypeptide fragments results in decreasedtriglyceride levels, decreased free fatty acid levels, decreased glucoselevels, and decreased body weight as well as increased muscle oxidation.

The invention is drawn inter alia to methods of preventing or treatingobesity-related diseases and disorders comprising providing anindividual in need of such treatment with an OBG3 or gOBG3 polypeptidefragment of the invention. Preferably, the OBG3 polypeptide fragment hasobesity-related activity either in vitro or in vivo. Preferably the OBG3polypeptide fragment is provided to the individual in a pharmaceuticalcomposition that is preferably taken orally. Preferably the individualis a mammal, and most preferably a human. In preferred embodiments, theobesity-related disease or disorder is selected from the groupconsisting of atherosclerosis, cardiovascular disease, impaired glucosetolerance, insulin resistance, hypertension, stroke, Syndrome X, Type Idiabetes, Type II diabetes and lhpoatrophic diabetes. Diabetes-relatedcomplications to be treated by the methods of the invention includemicroangiopathic lesions, ocular lesions, retinopathy, neuropathy andrenal lesions. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Otherobesity-related disorders to be treated by compounds of the inventioninclude hyperlipidemia, hypertriglyceridemia, and hyperuricemia. Yetother obesity-related diseases or disorders of the invention includecachexia, wasting, AIDS-related weight loss, neoplasia-related weightloss, anorexia, and bulimia. In highly preferred embodiments, OBG3polypeptide fragments in pharmaceutical compositions are used tomodulate body weight in healthy individuals for cosmetic reasons.

The invention also features a method of preventing or treatingobesity-related diseases and disorders comprising providing anindividual in need of such treatment with a compound identified byassays of the invention (described in Section VI of the PreferredEmbodiments of the Invention and in the Examples). Preferably thesecompounds antagonize or agonize effects of OBG3 or gOBG3 polypeptidefragments in cells in vitro, muscles ex vivo, or in animal models.Alternatively, these compounds agonize or antagonize the effects of OBG3or gOBG3 polypeptide fragments on leptin and/or lipoprotein uptakeand/or binding. Optionally, these compounds prevent the interaction,binding, or uptake of OBG3 or gOBG3 polypeptide fragments with LSR invitro or in vivo. Preferably, the compound is provided to the individualin a pharmaceutical composition that is preferably taken orally.Preferably the individual is a mammal, and most preferably a human. Inpreferred embodiments, the obesity-related disease or disorder isselected from the group consisting of obesity and obesity-relateddiseases and disorders such as atherosclerosis, heart disease, impairedglucose tolerance, insulin resistance, hypertension, stroke Syndrome X,Type I diabetes, Type II diabetes, and lipoatrophic diabetes.Diabetes-related complications to be treated by the methods of theinvention include microangiopathic lesions, ocular lesions, retinopathy,neuropathy and renal lesions. Heart disease includes, but is not limitedto, cardiac insufficiency, coronary insufficiency, and high bloodpressure. Other obesity-related disorders to be treated by compounds ofthe invention include hyperlipidemia, hypertriglyceridemia andhyperuricemia. Yet other obesity-related diseases or disorders of theinvention include cachexia, wasting, AIDS-related weight loss,neoplasia-related weight loss, anorexia, and bulimia. In highlypreferred embodiments, the pharmaceutical compositions are used tomodulate body weight for cosmetic reasons.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance, incombination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some individuals, particularly thosewith Type I diabetes, Type II diabetes, or insulin resistance, incombination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance,alone, without combination of insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some individuals, particularly thosewith Type II diabetes or insulin resistance, alone, without combinationof insulin therapy.

In a further preferred embodiment, the present invention may be used incomplementary therapy, particularly in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance, toimprove their weight or glucose control in combination with an oralinsulin secretagogue or an insulin sensitising agent. Preferably, theoral insulin secretagogue is 1,1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected fromtolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride,glipizide and glidazide. Preferably, the insulin sensitising agent isselected from metformin, ciglitazone, toglitazone and pioglitazone.

The present invention further provides a method of improving the bodyweight or glucose control of some individuals, particularly those withType I diabetes. Type II diabetes, or insulin resistance, alone, withoutan oral insulin secretagogue or an insulin sensitising agent.

In a further preferred embodiment, the present invention may beadministered either concomitantly or concurrently, with the oral insulinsecretagoaue or insulin sensitising agent for example in the form ofseparate dosage units to be used simultaneously, separately orsequentially (either before or after the secretagogue or either beforeor after the sensitising agent). Accordingly, the present inventionfurther provides for a composition of pharmaceutical or physiologicallyacceptable composition and an oral insulin secretagogue or insulinsensitising agent as a combined preparation for simultaneous, separateor sequential use for the improvement of body weight or glucose controlin some individuals, particularly those with Type I diabetes, Type IIdiabetes, or insulin resistance.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition furtherprovides a method for the use as an insulin sensitiser.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some individuals,particularly those with Type I diabetes, Type II diabetes, or insulinresistance, in combination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some individuals,particularly those with Type II diabetes or insulin resistance, withoutinsulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition furtherprovides a method for the use as an inhibitor of the progression fromimpaired glucose tolerance to insulin resistance.

More generally, the instant invention is drawn to treatment with OBG3and gOBG3 polypeptide fragments where an individual is shown to have aparticular genotype for an APM1 marker (APM1 designates the humanhomolog of the full-length OBG3 polypeptide), or where they have beenshown to have a reduced amount of plasma APM1, either full-length orpreferably a more biologically active fragment of APM1, as compared tocontrol values, e.g., values representative of non-diseased individuals,or as compared to that individual prior to the onset of a disease orcondition, in either case, treatment comprises providingpharmaceutically acceptable gOBG3 or OBG3 polypeptide fragments to theindividual. The exact amount of OBG3 or gOBG3 fragment provided would bedetermined through clinical trials under the guidance of qualifiedphysicians, but would be expected to be in the range of 5-7 mg perindividual per day. In general, a preferred range would be from 0.5 to14 mg per individual per day, with a highly preferred range beingbetween 1 and 10 mg per individual per day. Individuals who couldbenefit from treatment with gOBG3 or OBG3 polypeptide fragments could beidentified through at least two methods: plasma serum leveldeterminations and genotyping.

OBG3/APM1 Levels

Preliminary studies have shown that obese people have lower levels offull-length OBG3/APM1 than non-obese people. The invention envisionstreatment of individuals (preferably obese) that have low levels offull-length OBG3/APM1 with OBG3 or gOBG3 polypeptide fragments of theinvention. In addition, the invention preferably is drawn to treatmentof individuals with low levels of the biologically active fragment ofOBG3/APM1 with OBG3 or gOBG3 polypeptide fragments of the invention. Infurther embodiments, OBG3 or gOBG3 polypeptide fragments of the presentinvention are administered to individuals, preferably obese individuals,that levels of full-length OBG3 (or alternatively a mature OBG3polypeptide fragment) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, about 100% or 100% lower than non-obese individuals, preferablyhealthy individuals as determined by a physician using normal standardsin the art. Methods to determine and compare the levels of full-lengthOBG3 in individuals are well-known in the art and include, but are notlimited to using an antibody specific for APM1 in a format such as aRadio Immune Assay, ELISA, Western blot, dotblot, or as part of anarray, for example. Methods of generating antibodies to, and detectionof, APM1 and fragments thereof (such as the 27 kDa APM1 product in humanplasma) as well as to proteins with SNPs are included in the presentinvention and are discussed in PCT/IB99/01858, U.S. application Ser. No.09/434,848 and WO 99/07736, hereby incorporated herein by reference inits entirety including drawings, figures, or tables. Further, antibodiesspecific for OBG3/gOBG3 polypeptide fragments of the invention (e.g.,antibodies that bind the 27 kDa APM1 fragment as well as full-lengthAPM1 and antibodies that bind the 27 kDa APM1 fragment but not thefull-length APM1), their generation, and their use are described herein.Particularly included in the present invention are methods of binding anantibody specific for the 27 kDa APM1 fragment to the 27 kDa APM1fragment comprising the steps of: obtaining a biological samplecomprising the 27 kDa APM1 fragment, contacting said 27 kDa fragmentwith said antibody under conditions that allow specific binding of theantibody and the 27 kDa fragment to occur, and specifically binding saidantibody to said 27 kDa fragment.

Also particularly included in the present invention are monoclonalantibodies that specifically bind an APM1 polypeptide fragment comprisedof the C-terminal globular domain (gAPM1) but not said fragment fromwhich the N-terminal dipeptide EP has been proteolytically removed bydipeptidyl peptidase [Deacon (2000) Journal of Clinical Endocrinologyand Metabolism 85:3575-3581 which disclosure is hereby incorporated byreference in its entirety]. Preferred said gAPM1 polypeptide fragment isselected from amino acids 85-244 or 103-244 of SEQ ID NO: 6. Methods ofmaking monoclonal antibodies are known to those of ordinary skill in theart. Further particularly included are methods of using gAPM1 of theinvention to select monoclonal antibodies, wherein said monoclonalantibody specifically binds to gAPM1 (85-244) but not to gAPM1 (87-244)of SEQ ID NO:6 comprising the steps of: obtaining a sample comprisinggAPM1 (85-244) and obtaining a sample comprising gAPM1 (87-244);contacting said antibody with gAPM1 (85-244) and contacting saidantibody with gAPM1 (87-244); and quantifying the level of antibodybinding to gAPM1 (85-244) and the level of antibody binding to gAPM1(87-244) by enzyme-linked immunosorbent assay (ELISA). Furtherparticularly included are methods of selecting monoclonal antibodies,wherein said monoclonal antibody specifically binds to gAPM1 (103-247)but not to gAPM1 (105-247) of SEQ ID NO:2 or SEQ ID NO:4 comprising thesteps of: obtaining a sample comprising gAPM1 (103-247) and obtaining asample comprising gAPM1 (87-244); contacting said antibody with gAPM1(105-247) and contacting said antibody with gAPM1 (87-244); andquantifying the level of antibody binding to gAPM1 (103-247) and thelevel of antibody binding to gAPM1 (105-247) by ELISA.

APM1 Genotyping

The methods treatment using genotyping to identify individuals thatwould benefit from treatments of the invention are based on the findingthat single nucleotide polymorphisms (SNPs) in the APM1 gene have beenidentified that show an association in obese adolescents with free fattyacid (FFA) and respiratory quotient levels, others that show anassociation with the relationship between BMI and leptin, and stillothers that show an association with glucose levels. Further, acombination of the APM1 SNPs associated with FFA and leptin metabolismalso predicts people who will be seriously overweight (data not shown).

APM1 SNPs and methods of genotyping are described in PCT, IB99/01858 aswell as U.S. application Ser. No. 09/434,848, both of which are herebyincorporated herein in their entirety including any drawings, figure, ortables. Briefly, the term “genotype” as used herein refers to theidentity of the alleles present n an individual or a sample. The term“genotyping” a sample or an individual for a biallelic marker consistsof determining the specific allele or the specific nucleotide carried byan individual at a biallelic marker.

Methods of genotyping comprise determining the identity of a nucleotideat an APM1 biallelic marker site by any method known in the art.Preferably, microsequencing is used. The genotype is used to determinewhether an individual should be treated with gOBG3 or OBG3 polypeptidefragments. Thus, these genotyping methods are performed on nucleic acidsamples derived from a single individual. These methods are well-knownin the art, and discussed fully in the applications referenced above andbriefly below.

Any method known in the art can be used to identify the nucleotidepresent at a biallelic marker site. Since the biallelic marker allele tobe detected has been identified and specified in the present invention,detection will prove simple for one of ordinary skill in the art byemploying any of a number of techniques. Many genotyping methods requirethe previous amplification of the DNA region carrying the biallelicmarker of interest. While the amplification of target or signal is oftenpreferred at present, ultrasensitive detection methods that do notrequire amplification are also encompassed by the present genotypingmethods.

Methods well-known to those skilled in the art that can be used todetect biallelic polymorphisms include methods such as conventional dotblot analysis, single strand conformational polymorphism analysis (SSCP:Orita et al. (1989) Proc Natl Acad Sci USA 86(8):2766-70), denaturinggradient gel electrophoresis (DGGE), heteroduplex analysis, mismatchcleavage detection, and other conventional techniques as described inSheffield et al. (1991; Am J Hum Genet. 49(4):699-706); White et al.(1992). Grompe et al. ((1989) Proc Natl Acad Sci USA 86(15):5888-92;(1993) Nat Genet. 5(2):111-7). Another method for determining theidentity of the nucleotide present at a particular polymorphic siteemploys a specialized exonuclease-resistant nucleotide derivative asdescribed in U.S. Pat. No. 4,656,127. Preferred methods involve directlydetermining the identity of the nucleotide present at a biallelic markersite by sequencing assay, allele-specific amplification assay, orhybridization assay. The following is a description of some preferredmethods. A highly preferred method is the microsequencing technique. Theterm “sequencing” is used herein to refer to polymerase extension ofduplex primer/template complexes and includes both traditionalsequencing and microsequencing.

1) Sequencing Assays

The nucleotide present at a polymorphic site can be determined bysequencing methods. In a preferred embodiment. DNA samples are subjectedto PCR amplification before sequencing using any method known in theart. Preferably, the amplified DNA is subjected to automated dideoxyterminator sequencing reactions using a dye-primer cycle sequencingprotocol. Sequence analysis allows the identification of the basepresent at the biallelic marker site.

2) Microsequencing Assays

In microsequencing methods, the nucleotide at a polymorphic site in atarget DNA is detected by a single nucleotide primer extension reaction.This method involves appropriate microsequencing primers that hybridizejust upstream of the polymorphic base of interest in the target nucleicacid. A polymerase is used to specifically extend the 3′ end of theprimer with one single ddNTP (chain terminator) complementary to thenucleotide at the polymorphic site. The identity of the incorporatednucleotide is then determined in any suitable way.

Typically, microsequencing reactions are carried out using fluorescentddNTPs and the extended microsequencing primers are analyzed byelectrophoresis on ABI 377 sequencing machines to determine the identityof the incorporated nucleotide as described in EP 412 883. Alternativelycapillary electrophoresis can be used in order to process a highernumber of assays simultaneously.

Different approaches can be used for the labeling and detection ofddNTPs. A homogeneous phase detection method based on fluorescenceresonance energy transfer has been described by Chen and Kwok ((1997)Nucleic Acids Res 25(2):347-53) and Chen et al. ((1997) Proc Natl AcadSci USA 94(20):10756-61). In this method, amplified genomic DNAfragments containing polymorphic sites are incubated with a5′-fluorescein-labeled primer in the presence of allelic dye-labeleddideoxyribonucleoside triphosphates and a modified Taq polymerase. Thedye-labeled primer is extended one base by the dye-terminator specificfor the allele present on the template. At the end of the genotypingreaction, the fluorescence intensities of the two dyes in the reactionmixture are analyzed directly without separation or purification. Allthese steps can be performed in the same tube and the fluorescencechanges can be monitored in real time. Alternatively, the extendedprimer may be analyzed by MALDI-TOF Mass Spectrometry. The base at thepolymorphic site is identified by the mass added onto themicrosequencing primer (see Haff and Smirnov, (1997) Nucleic Acids Res25(18):3749-50: (1997) Genome Res 7(4):378-88).

Microsequencing may be achieved by the established microsequencingmethod or by developments or derivatives thereof. Alternative methodsinclude several solid-phase microsequencing techniques. The basicmicrosequencing protocol is the same as described previously, exceptthat the method is conducted as a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized or captured onto asolid support. To simplify the primer separation and the terminalnucleotide addition analysis, oligonucleotides are attached to solidsupports or are modified in such ways that permit affinity separation aswell as polymerase extension. The 5′ ends and internal nucleotides ofsynthetic oligonucleotides can be modified in a number of different waysto permit different affinity separation approaches, e.g., biotinylation.If a single affinity group is used on the oligonucleotides, theoligonucleotides can be separated from the incorporated terminatorregent. This eliminates the need of physical or size separation. Morethan one oligonucleotide can be separated from the terminator reagentand analyzed simultaneously if more than one affinity group is used.This permits the analysis of several nucleic acid species or morenucleic acid sequence information per extension reaction. The affinitygroup need not be on the priming oligonucleotide but could alternativelybe present on the template.

For example, immobilization can be carried out via an interactionbetween biotinylated DNA and streptavidin-coated microtitration wells oravidin-coated polystyrene particles. In the same manner,oligonucleotides or templates may be attached to a solid support in ahigh-density format. In such solid phase microsequencing reactions,incorporated ddNNTPs can be radiolabeled (Syvänen, (1994) Clin Chim Acta226(2):225-36) or linked to fluorescein (Livak and Hainer, (1994) HumMutat 3(4):379-85). The detection of radiolabeled ddNTPs can be achievedthrough scintillation-based techniques. The detection offluorescein-linked ddNTPs can be based on the binding of antifluoresceinantibody conjugated with alkaline phosphatase, followed by incubationwith a chromogenic substrate (such as p-nitrophenyl phosphate).

Other possible reporter-detection pairs include: ddNTP linked todinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harjuet al. (1993) Clin Chem 39(11 Pt 1):2282-7) or biotinylated ddNTP andhorseradish peroxidase-conjugated streptavidin with o-phenylenediamineas a substrate (WO 92/15712). As yet another alternative solid-phasemicrosequencing procedure. Nyren et al. ((1993) Anal Biochem208(1):171-5) described a method relying on the detection of DNApolymerase activity by an enzymatic luminometric inorganic pyrophosphatedetection assay (ELIDA).

Pastinen et al. ((1997) Genome Res 7(6):606-14) describe a method formultiplex detection of single nucleotide polymorphism in which the solidphase minisequencing principle is applied to an oligonucleotide arrayformat. High-density arrays of DNA probes attached to a solid support(DNA chips) are further described below.

It will be appreciated that any primer having a 3′ end immediatelyadjacent to the polymorphic nucleotide may be used. Similarly, it willbe appreciated that microsequencing analysis may be performed for anybiallelic marker or any combination of biallelic markers of the presentinvention.

3) Allele-Specific Amplification Assay Methods

Discrimination between the two alleles of a biallelic marker can also beachieved by allele specific amplification, a selective strategy, wherebyone of the alleles is amplified without, or at a much higher rate than,amplification of the other allele. This is accomplished by placing thepolymorphic base at the 3′ end of one of the amplification primers.Because the extension forms from the 3′ end of the primer, a mismatch ator near this position has an inhibitory effect on amplification.Therefore, under appropriate amplification conditions, these primersonly direct amplification on their complementary allele. Determining theprecise location of the mismatch and the corresponding assay conditionsare well with the ordinary skill in the art.

The “Oligonucleotide Ligation Assay” (OLA) uses two oligonucleotidesthat are designed to be capable of hybridizing to abutting sequences ofa single strand of a target molecule. One of the oligonucleotides isbiotinylated, and the other is detectably labeled, if the precisecomplementary sequence is found in a target molecule, theoligonucleotides will hybridize such that their termini abut, and createa ligation substrate that can be captured and detected. OLA is capableof detecting single nucleotide polymorphisms and may be advantageouslycombined with PCR as described by Nickerson et al. ((1990) Proc NatlAcad Sci USA 87(22):8923-7). In this method. PCR is used to achieve theexponential amplification of target DNA, which is then detected usingOLA.

Other amplification methods that are particularly suited for thedetection of single nucleotide polymorphism include LCR (ligase chainreaction) and Gap LCR (GLCR). LCR uses two pairs of probes toexponentially amplify a specific target. The sequences of each pair ofoligonucleotides are selected to permit the pair to hybridize toabutting sequences of the same strand of the target. Such hybridizationforms a substrate for a template-dependant ligase. In accordance withthe present invention, LCR can be performed with oligonucleotides havingthe proximal and distal sequences of the same strand of a biallelicmarker site.

In one embodiment, either oligonucleotide will be designed to includethe biallelic marker site. In such an embodiment, the reactionconditions are selected such that the oligonucleotides can be ligatedtogether only if the target molecule either contains or lacks thespecific nucleotide that is complementary to the biallelic marker on theoligonucleotide.

In an alternative embodiment, the oligonucleotides will not include thebiallelic marker, such that when they hybridize to the target molecule,a “gap” is created as described in WO 90:01069. This gap is then“filled” with complementary dNTPs (as mediated by DNA polymerase), or byan additional pair of oligonucleotides. Thus at the end of each cycle,each single strand has a complement capable of serving as a targetduring the next cycle and exponential allele-specific amplification ofthe desired sequence is obtained. Ligase/Polymerase-mediated Genetic BitAnalysis™ is another method for determining the identity of a nucleotideat a preselected site in a nucleic acid molecule (WO 95/21271). Thismethod involves the incorporation of a nucleoside triphosphate that iscomplementary to the nucleotide present at the preselected site onto theterminus of a primer molecule, and their subsequent ligation to a secondoligonucleotide. The reaction is monitored by detecting a specific labelattached to the reaction's solid phase or by detection in solution.

4) Hybridization Assay Methods

A preferred method of determining the identity of the nucleotide presentat a biallelic marker site involves nucleic acid hybridization. Thehybridization probes, which can be conveniently used in such reactions,preferably include probes specific for APM1 cDNA surrounding APM1biallelic markers. Any hybridization assay may be used includingSouthern hybridization, Northern hybridization, dot blot hybridizationand solid-phase hybridization (see Sambrook et al., supra).

Hybridization refers to the formation of a duplex structure by twosingle stranded nucleic acids due to complementary base pairing.Hybridization can occur between exactly complementary nucleic acidstrands or between nucleic acid strands that contain minor regions ofmismatch. Specific probes can be designed that hybridize to one form ofa biallelic marker and not to the other and therefore are able todiscriminate between different allelic forms. Allele-specific probes areoften used in pairs, one member of a pair showing perfect match to atarget sequence containing the original allele and the other showing aperfect match to the target sequence containing the alternative allele.

Hybridization conditions should be sufficiently stringent that there isa significant difference in hybridization intensity between alleles, andpreferably an essentially binary response, whereby a probe hybridizes toonly one of the alleles. Stringent, sequence specific hybridizationconditions, under which a probe will hybridize only to the exactlycomplementary target sequence are well known in the art (Sambrook etal., supra). Stringent conditions are sequence dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 5° C., lower than the thermal melting point(Tm) for the specific sequence at a defined ionic strength and pH.Although such hybridizations can be performed in solution, it ispreferred to employ a solid-phase hybridization assay. The target DNAcomprising a biallelic marker of the present invention may be amplifiedprior to the hybridization reaction.

The presence of a specific allele in the sample is determined bydetecting the presence or the absence of stable hybrid duplexes formedbetween the probe and the target DNA. The detection of hybrid duplexescan be carried out by a number of methods. Various detection assayformats are well known which utilize detectable labels bound to eitherthe target or the probe to enable detection of the hybrid duplexes.Typically, hybridization duplexes are separated from unhybridizednucleic acids and the labels bound to the duplexes are then detected.Those skilled in the art will recognize that wash steps may be employedto wash away excess target DNA or probe as well as unbound conjugate.Further, standard heterogeneous assay formats are suitable for detectingthe hybrids using the labels present on the primers and probes.

Two recently developed assays allow hybridization-based allelediscrimination with no need for separations or washes (see Landegren U,et al., (1998) Genome Res 8(8):769-76). The TaqMan assay takes advantageof the 5′ nuclease activity of Taq DNA polymerase to digest a DNA probeannealed specifically to the accumulating amplification product. TaqManprobes are labeled with a donor-acceptor dye pair that interacts viafluorescence resonance energy transfer (FRET). Cleavage of the TaqManprobe by the advancing polymerase during amplification dissociates thedonor dye from the quenching acceptor dye, greatly increasing the donorfluorescence. All reagents necessary to detect two allelic variants canbe assembled at the beginning of the reaction and the results aremonitored in real time (see Livak et al., 1995).

In an alternative homogeneous hybridization based procedure, molecularbeacons are used for allele discriminations. Molecular beacons arehairpin-shaped oligonucleotide probes that report the presence ofspecific nucleic acids in homogeneous solutions. When they bind to theirtargets they undergo a conformational reorganization that restores thefluorescence of an internally quenched fluorophore (Tyagi et al., (1998)Nat Biotechnol 16(1):49-53).

The polynucleotides provided herein can be used to produce probes thatcan be used in hybridization assays for the detection of biallelicmarker alleles in biological samples. These probes are characterized inthat they preferably comprise between 8 and 50 nucleotides, and in thatthey are sufficiently complementary to a sequence comprising a biallelicmarker of the present invention to hybridize thereto and preferablysufficiently specific to be able to discriminate the targeted sequencefor only one nucleotide variation. A particularly preferred probe is 25nucleotides in length. Preferably the biallelic marker is within 4nucleotides of the center of the polynucleotide probe. In particularlypreferred probes, the biallelic marker is at the center of saidpolynucleotide. In preferred embodiments the polymorphic base is within5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide,more preferably at the center of said polynucleotide. Preferably theprobes of the present invention are labeled or immobilized on a solidsupport.

By assaying the hybridization to an allele specific probe, one candetect the presence or absence of a biallelic marker allele in a givensample. High-Throughput parallel hybridizations in array format arespecifically encompassed within “hybridization assays” and are describedbelow.

5) Hybridization to Addressable Arrays of Oliionucleotides

Hybridization assays based on oligonucleotide arrays rely on thedifferences in hybridization stability of short oligonucleotides toperfectly matched and mismatched target sequence variants. Efficientaccess to polymorphism information is obtained through a basic structurecomprising high-density arrays of oligonucleotide probes attached to asolid support (e.g., the chip) at selected positions. Each DNA chip cancontain thousands to millions of individual synthetic DNA probesarranged in a grid-like pattern and miniaturized to the size of a dime.

The chip technology has already been applied with success in numerouscases. For example, the screening of mutations has been undertaken inthe BRCA1 gene, in S. cerevisiae mutant strains, and in the proteasegene of HIV-1 virus (Hacia et al., (1996) Nat Genet. 14(4):441-7:Shoemaker et al., (1996) Nat Genet. 14(4):450-6; Kozal et al., (1996)Nat Med 2(7):753-9). Chips of various formats for use in detectingbiallelic polymorphisms can be produced on a customized basis byAffymetrix (GeneChip™), Hyseq (HyChip and HyGnostics), and ProtogeneLaboratories.

In general, these methods employ arrays of oligonucleotide probes thatare complementary to target nucleic acid sequence segments from anindividual, which target sequences include a polymorphic marker. EP785280 describes a tiling strategy for the detection of singlenucleotide polymorphisms.

Briefly, arrays may generally be “tiled” for a large number of specificpolymorphisms. By “tiling” is generally meant the synthesis of a definedset of oligonucleotide probes which is made up of a sequencecomplementary to the target sequence of interest, as well as preselectedvariations of that sequence, e.g., substitution of one or more givenpositions with one or more members of the basis set of monomers, i.e.,nucleotides. Tiling strategies are further described in PCT applicationNo. WO 95/11995. In a particular aspect, arrays are tiled for a numberof specific, identified biallelic marker sequences. In particular, thearray is tiled to include a number of detection blocks, each detectionblock being specific for a specific biallelic marker or a set ofbiallelic markers.

For example, a detection block may be tiled to include a number ofprobes, which span the sequence segment that includes a specificpolymorphism. To ensure probes that are complementary to each allele,the probes are synthesized in pairs differing at the biallelic marker.In addition to the probes differing at the polymorphic base,monosubstituted probes are also generally tiled within the detectionblock. These monosubstituted probes have bases at and up to a certainnumber of bases in either direction from the polymorphism, substitutedwith the remaining nucleotides (selected from A, T, G, C and U).Typically the probes in a tiled detection block will includesubstitutions of the sequence positions up to and including those thatare 5 bases away from the biallelic marker. The monosubstituted probesprovide internal controls for the tiled array, to distinguish actualhybridization from artifactual cross-hybridization. Upon completion ofhybridization with the target sequence and washing of the array, thearray is scanned to determine the position on the array to which thetarget sequence hybridizes. The hybridization data from the scannedarray is then analyzed to identify, which allele or alleles of thebiallelic marker are present in the sample. Hybridization and scanningmay be carried out as described in PCT application No. WO 92/10092 andWO 95/11995 and U.S. Pat. No. 5,424,186.

Thus, in some embodiments, the chips may comprise an array of nucleicacid sequences of fragments of about 15 nucleotides in length. Inpreferred embodiments the polymorphic base is within 5, 4, 3, 2, 1,nucleotides of the center of the said polynucleotide, more preferably atthe center of said polynucleotide. In some embodiments, the chip maycomprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of thesepolynucleotides.

6) Integrated Systems

Another technique, which may be used to analyze polymorphisms, includesmulticomponent integrated systems, which miniaturize andcompartmentalize processes such as PCR and capillary electrophoresisreactions in a single functional device. An example of such technique isdisclosed in U.S. Pat. No. 5,589,136, which describes the integration ofPCR amplification and capillary electrophoresis in chips.

Integrated systems can be envisaged mainly when microfluidic systems areused. These systems comprise a pattern of microchannels designed onto aglass, silicon, quartz, or plastic wafer included on a microchip. Themovements of the samples are controlled by electric, electroosmotic orhydrostatic forces applied across different areas of the microchip tocreate functional microscopic valves and pumps with no moving parts.Varying the voltage controls the liquid flow at intersections betweenthe micro-machined channels and changes the liquid flow rate for pumpingacross different sections of the microchip.

For genotyping biallelic markers, the microfluidic system may integratenucleic acid amplification, microsequencing, capillary electrophoresisand a detection method such as laser-induced fluorescence detection.

In a first step, the DNA samples are amplified, preferably by PCR. Then,the amplification products are subjected to automated microsequencingreactions using ddNTPs (specific fluorescence for each ddNTP) and theappropriate oligonucleotide microsequencing primers which hybridize justupstream of the targeted polymorphic base. Once the extension at the 3′end is completed, the primers are separated from the unincorporatedfluorescent ddlNTPs by capillary electrophoresis. The separation mediumused in capillary electrophoresis can for example be polyacrylamide,polyethyleneglycol or dextran. The incorporated ddNTPs in thesingle-nucleotide primer extension products are identified byfluorescence detection. This microchip can be used to process at least96 to 384 samples in parallel. It can use the usual four-color laserinduced fluorescence detection of the ddNTPs.

APM1 Biallellic Markers

The APM 1 biallelic markers currently identified are shown in Table 1below. The markers that have been linked with either FFA levels orchanges in the leptin/BMI index are A5, A6, A7 and A3, A4, respectively.A5, A6, and A7 are in complete linkage disequilibrium. Thus, if anindividual's genotype at A6 is GG, then A7 will be AA, and both arelinked with decreased FFA levels and would indicate that treatment withgOBG3 or OBG3 polypeptide fragments was appropriate for example.Similarly, if an individual's genotype at A4 is AC or CC, treatment withgOBG3 or OBG3 polypeptide fragments could be expected to be beneficial.Alternatively, if an individual has both an AA genotype at A7 and an ACor CC genotype at A4, treatment with gOBG3 or OBG3 polypeptide fragmentsis indicated.

The above-described associations between genotypes and risk factors andtreatment are exemplary, only. Other associations that would alsoindicate individuals appropriate for gOBG3 or OBG3 fragment treatment(or inappropriate) can also be identified using the methods described inthe art or PCT/IB99/01858. Associations that would indicate treatmentwould be those genotypes associated with changes in parameters thatgOBG3 or OBG3 fragment administration has been shown to affect in a“positive” direction, e.g., the association with decrease in weight fortreatment of obesity. Associations that would indicate that treatmentshould not be performed would be genotypes that indicated an adverseaffect for diabetes treatment (negative effect on insulin levels forexample) or weight loss.

TABLE 1 Biallelic Marker Localization in Polymor- Marker positionAmplicon marker Name APM1 gene phism in SEQ ID No7 9-27 A1 9-27/261 5′regulatory Allele 1: G 3787 region Allele 2: C 99-14387 A2 99-14387 129Intron 1 Allele 1: A 11118 Allele 2: C 9-12 A3 9-12/48 Intron 1 Allele1: T 15120 Allele 2: C 9-12 and A4 9-12/124 or Exon 2 Allele 1: T 151969-13 9-13/66 Allele 2: G 9-12 and A5 9-12/355 or Intron 2 Allele 1: G15427 9-13 9-13/297 Allele 2: T 9-12 and A6 9-12/428 or Intron 2 Allele1: A 15500 9-13 9-13/370 Allele 2: G 99-14405 A7 99-14405/105 Intron 2Allele 1: G 15863 Allele 2: A 9-16 A8 9-16/189 Exon 3 Allele 1: A 17170Allele 2: Del

APM1 Association Studies

Association studies focus on population frequencies and rely on thephenomenon of linkage disequilibrium. Linkage disequilibrium is thedeviation from random of the occurrence of pairs of specific alleles atdifferent loci on the same chromosome. If a specific allele in a givengene is directly associated with a particular trait, its frequency willbe statistically increased in an affected (trait positive) population,when compared to the frequency in a trait negative population or in arandom control population. As a consequence of the existence of linkagedisequilibrium, the frequency of all other alleles present in thehaplotype carrying the trait-causing allele will also be increased intrait positive individuals compared to trait negative individuals orrandom controls. Therefore, association between the trait and any allele(specifically a biallelic marker allele) in linkage disequilibrium withthe trait-causing allele will suffice to suggest the presence of atrait-related gene in that particular region.

Case-control populations can be genotyped for biallelic markers toidentify associations that narrowly locate a trait causing allele, asany marker in linkage disequilibrium with one given marker associatedwith a trait will be associated with the trait. Linkage disequilibriumallows the relative frequencies in case-control populations of a limitednumber of genetic polymorphisms (specifically biallelic markers) to beanalyzed as an alternative to screening all possible functionalpolymorphisms in order to find trait-causing alleles. Associationstudies compare the frequency of marker alleles in unrelatedcase-control populations, and represent powerful tools for thedissection of complex traits.

Case-Control Populations (Inclusion Criteria)

Population-based association studies do not concern familialinheritance, but compare the prevalence of a particular genetic marker,or a set of markers, in case-control populations. They are case-controlstudies based on comparison of unrelated case (affected or traitpositive) individuals and unrelated control (unaffected, trait negativeor random) individuals. Preferably, the control group is composed ofunaffected or trait negative individuals. Further, the control group isethnically matched to the case population. Moreover, the control groupis preferably matched to the case-population for the main knownconfusion factor for the trait under study (for example age-matched foran age-dependent trait). Ideally, individuals in the two samples arepaired in such a way that they are expected to differ only in theirdisease status. The terms “trait positive population”, “case population”and “affected population” are used interchangeably herein.

An important step in the dissection of complex traits using associationstudies is the choice of case-control populations (see, Lander andSchork, (1994) Science 265(5181):2037-48). A major step in the choice ofcase-control populations is the clinical definition of a given trait orphenotype. Any genetic trait may be analyzed by the association methodproposed here by carefully selecting the individuals to be included inthe trait positive and trait negative phenotypic groups. Four criteriaare often useful: clinical phenotype, age at onset, family history andseverity.

The selection procedure for continuous or quantitative traits (such asblood pressure for example) involves selecting individuals at oppositeends of the phenotype distribution of the trait under study, so as toinclude in these trait positive and trait negative populationsindividuals with non-overlapping phenotypes. Preferably, case-controlpopulations consist of phenotypically homogeneous populations. Traitpositive and trait negative populations consist of phenotypicallyuniform populations of individuals representing each between 1 and 98%,preferably between 1 and 80%, more preferably between 1 and 50%, andmore preferably between 1 and 30%, most preferably between 1 and 20% ofthe total population under study, and preferably selected amongindividuals exhibiting non-overlapping phenotypes. The clearer thedifference between the two trait phenotypes, the greater the probabilityof detecting an association with biallelic markers. The selection ofthose drastically different but relatively uniform phenotypes enablesefficient comparisons in association studies and the possible detectionof marked differences at the genetic level, provided that the samplesizes of the populations under study are significant enough.

In preferred embodiments, a first group of between 50 and 300 traitpositive individuals, preferably about 100 individuals, are recruitedaccording to their phenotypes. A similar number of trait negativeindividuals are included in such studies.

In the present invention, typical examples of inclusion criteria includeobesity and disorders related to obesity as well as physiologicparameters associated with obesity, such as free fatty acid levels,glucose levels, insulin levels, leptin levels, triglyceride levels, freefatty acid oxidation levels, and weight loss.

Association Analysis

The general strategy to perform association studies using biallelicmarkers derived from a region carrying a candidate gene is to scan twogroups of individuals (case-control populations) in order to measure andstatistically compare the allele frequencies of the biallelic markers ofthe present invention in both groups.

If a statistically significant association with a trait is identifiedfor at least one or more of the analyzed biallelic markers, one canassume that: either the associated allele is directly responsible forcausing the trait (i.e., the associated allele is the trait causingallele), or more likely the associated allele is in linkagedisequilibrium with the trait causing allele. The specificcharacteristics of the associated allele with respect to the candidategene function usually give further insight into the relationship betweenthe associated allele and the trait (causal or in linkagedisequilibrium). If the evidence indicates that the associated allelewithin the candidate gene is most probably not the trait-causing allelebut is in linkage disequilibrium with the real trait-causing allele,then the trait-causing allele can be found by sequencing the vicinity ofthe associated marker, and performing further association studies withthe polymorphisms that are revealed in an iterative manner.

Association studies are usually run in two successive steps. In a firstphase, the frequencies of a reduced number of biallelic markers from thecandidate gene are determined in the trait positive and trait negativepopulations. In a second phase of the analysis, the position of thegenetic loci responsible for the given trait is further refined using ahigher density of markers from the relevant region. However, if thecandidate gene under study is relatively small in length, as is the casefor APM1, a single phase may be sufficient to establish significantassociations.

Haplotype Analysis

As described above, when a chromosome carrying a disease allele firstappears in a population as a result of either mutation or migration, themutant allele necessarily resides on a chromosome having a set of linkedmarkers: the ancestral haplotype. This haplotype can be tracked throughpopulations and its statistical association with a given trait can beanalyzed. Complementing single point (allelic) association studies withmulti-point association studies also called haplotype studies increasesthe statistical power of association studies. Thus, a haplotypeassociation study allows one to define the frequency and the type of theancestral carrier haplotype. A haplotype analysis is important in thatit increases the statistical power of an analysis involving individualmarkers.

In a first stage of a haplotype frequency analysis, the frequency of thepossible haplotypes based on various combinations of the identifiedbiallelic markers of the invention is determined. The haplotypefrequency is then compared for distinct populations of trait positiveand control individuals. The number of trait positive individuals, whichshould be, subjected to this analysis to obtain statisticallysignificant results usually ranges between 30 and 300, with a preferrednumber of individuals ranging between 50 and 150. The sameconsiderations apply to the number of unaffected individuals (or randomcontrol) used in the study. The results of this first analysis providehaplotype frequencies in case-control populations, for each evaluatedhaplotype frequency a p-value and an odds ratio are calculated. If astatistically significant association is found the relative risk for anindividual carrying the given haplotype of being affected with the traitunder study can be approximated.

Interaction Analysis

The biallelic markers of the present invention may also be used toidentify patterns of biallelic markers associated with detectable traitsresulting from polygenic interactions. The analysis of geneticinteraction between alleles at unlinked loci requires individualgenotyping using the techniques described herein. The analysis ofallelic interaction among a selected set of biallelic markers withappropriate level of statistical significance can be considered as ahaplotype analysis. Interaction analysis consists in stratifying thecase-control populations with respect to a given haplotype for the firstloci and performing a haplotype analysis with the second loci with eachsubpopulation.

VII. Assays for Identifying Modulators of OBG3 Polypeptide FragmentActivity

The invention features methods of screening for one or more compoundsthat modulate OBG3 or gOBG3 polypeptide fragment activity in cells, thatincludes providing potential compounds to be tested to the cells, andwhere modulation of an OBG3 polypeptide fragment effect or activityindicates the one or more compounds. Exemplary assays that may be usedare described in the Examples 4-5, 7-14, 16, and 18. To these assayswould be added compounds to be tested for their inhibitory orstimulatory activity as compared to the effects of OBG3 polypeptidefragment alone. Other assays in which an effect is observed based on theaddition of OBG3 polypeptide fragment can also be used to screen formodulators of OBG3 polypeptide fragment activity or effects of thepresence of OBG3 polypeptide fragment on cells. The essential step is toapply an unknown compound and then to monitor an assay for a change fromwhat is seen when only OBG3 polypeptide fragment is applied to the cell.A change is defined as something that is significantly different in thepresence of the compound plus OBG3 polypeptide fragment compared to OBG3polypeptide fragment alone. In this case, significantly different wouldbe an “increase” or a “decrease” in a measurable effect of at least 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.

The term “modulation” as used herein refers to a measurable change in anactivity. Examples include, but are not limited to, lipolysis stimulatedreceptor (LSR) modulation, leptin modulation, lipoprotein modulation,plasma FFA levels, FFA oxidation, TG levels, glucose levels, and weight.These effects can be in vitro or preferably in vivo. Modulation of anactivity can be either an increase or a decrease in the activity. Thus,LSR activity can be increased or decreased, leptin activity can beincreased or decreased, and lipoprotein activity can be increased ordecreased. Similarly, FFA, TG, and glucose levels (and weight) can beincreased or decreased in vivo. Free Fatty Acid oxidation can beincreased or decreased in vivo or ex vivo.

By “LSR” activity is meant expression of LSR on the surface of the cell,or in a particular conformation, as well as its ability to bind, uptake,and degrade leptin and lipoprotein. By “leptin” activity is meant itsbinding, uptake and degradation by LSR, as well as its transport acrossa blood brain barrier, and potentially these occurrences where LSR isnot necessarily the mediating factor or the only mediating factor.Similarly, by “lipoprotein” activity is meant its binding, uptake anddegradation by LSR, as well as these occurrences where LSR is notnecessarily the mediating factor or the only mediating factor. Exemplaryassays are provided in Example 4-5, 7-14, 16, and 18. These assay andother comparable assays can be used to determine/identify compounds thatmodulate OBG3 polypeptide fragment activity. In some cases it may beimportant to identify compounds that modulate some but not all of theOBG3 polypeptide fragment activities, although preferably all activitiesare modified.

The term “increasing” as used herein refers to the ability of a compoundto increase an OBG3 polypeptide fragment activity in some measurable waycompared to the effect of an OBG3 polypeptide fragment in its absence.As a result of the presence of the compound leptin binding and/or uptakemight increase, for example, as compared to controls in the presence ofthe OBG3 polypeptide fragment alone. Preferably, an increase in activityis at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%compared to the level of activity in the presence of the OBG3 fragment.

Similarly, the term “decreasing” as used herein refers to the ability ofa compound to decrease an activity in some measurable way compared tothe effect of an OBG3 fragment in its absence. For example, the presenceof the compound decreases the plasma concentrations of FFA, TG, andglucose in mice. Also as a result of the presence of a compound leptinbinding and/or uptake might decrease, for example, as compared tocontrols in the presence of the OBG3 fragment alone. Preferably, adecrease in activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, or 75% as compared to the level of activity in the presence ofthe OBG3 fragment alone.

The invention features a method for identifying a potential compound tomodulate body mass in individuals in need of modulating body masscomprising: a) contacting a cell with a gOBG3 fragment and a candidatecompound; b) detecting a result selected from the group consisting ofLSR modulation, leptin modulation, lipoprotein modulation, FFA oxidationmodulation: and c) wherein said result identifies said potentialcompound if said result differs from said result when said cell iscontacted with the gOBG3 polypeptide fragment alone.

In preferred embodiments, said contacting further comprises a ligand ofsaid LSR. Preferably said ligand is selected from the group consistingof cytokine, lipoprotein, free fatty acids, and Clq, and more preferablysaid cytokine is leptin, and most preferably said leptin is a leptinpolypeptide fragment as described in U.S. Provisional application No.60/155,506 hereby incorporated by reference herein in its entiretyincluding any figures, drawings, or tables.

In other preferred embodiments, said OBG3 or gOBG3 polypeptide fragmentis mouse or is human. In other preferred embodiments, said cell isselected from the group consisting of PLC, CHO-K1, Hep3B, and HepG2.

In yet other preferred embodiments, said lipoprotein modulation isselected from the group consisting of binding, uptake, and degradation.Preferably, said modulation is an increase in said binding, uptake, ordegradation. Alternatively, said modulation is a decrease in saidbinding, uptake, or degradation.

In other preferred embodiments, leptin modulation is selected from thegroup consisting of binding, uptake, degradation, and transport.Preferably, said modulation is an increase in said binding, uptake,degradation, or transport. Alternatively, said modulation is a decreasein said binding, uptake, degradation, or transport. Preferably, saidtransport is across a blood-brain barrier.

In yet other preferred embodiments, said LSR modulation is expression onthe surface of said cell. Preferably, said detecting comprises FACS,more preferably said detecting further comprises antibodies that bindspecifically to said LSR, and most preferably said antibodies bindspecifically to the carboxy terminus of said LSR.

In still other preferred embodiments, said potential compound isselected from the group consisting of peptides, peptide libraries,non-peptide libraries, peptoids, fatty acids, lipoproteins, medicaments,antibodies, small molecules, and proteases. Other characteristics andadvantages of the invention are described in the Brief Description ofthe Figures and the Examples. These are meant to be exemplary only, andnot to limit the invention in any way. Throughout this application,various publications, patents and published patent applications arecited. The disclosures of these publications, patents and publishedpatent specifications referenced in this application are herebyincorporated by reference into the present disclosure.

VIII. Assays for Identifying Antagonists of Dipeptidyl PeptidaseCleavage of gOBG3 Polypeptide Fragment

The invention features methods of screening for one or more antagonistcompounds that block N-terminal cleavage of gOBG3 polypeptide fragmentby dipeptidyl peptidase. Preferred said compound is selected from but isnot restricted to small molecular weight organic or inorganic compound,protein, peptide, carbohydrate, or lipid. Preferred said dipeptidylpeptidase is human plasma comprised of dipeptidyl peptidase. Preferreddipeptidyl peptidase is selected from but not restricted to human CD26or human Attractin. Further preferred dipeptidyl peptidase is selectedfrom but is not restricted to soluble human CD26 or soluble humanAttractin. Soluble human CD26 is produced by recombinant means usingmethods well known to those of ordinary skill in the art (U.S. Pat. No.6,265,551 which disclosure is hereby incorporated by reference in itsentirety). Soluble human Attractin is produced by recombinant meansusing methods well known to those of ordinary skill in the art(International Patent Application No. WO15651A1 which disclosure ishereby incorporated by reference in its entirety). Preferred said gOBG3polypeptide fragment is selected from gOBG3 (85-244) or gOBG3 (103-244)of SEQ ID NO:6. Said dipeptidyl peptidase cleavage of said preferredgOBG3 polypeptide fragment (85-244) of SEQ ID NO:6 removes theN-terminal dipeptide VP. Said dipeptidyl peptidase cleavage of saidpreferred gOBG3 polypeptide fragment (103-244) of SEQ ID NO:6 removesthe N-terminal dipeptide EP. An exemplary assay that may be used isdescribed in Example 19.

Said methods of screening for an antagonist compound that blocksN-terminal cleavage of gOBG3 polypeptide fragment by dipeptidylpeptidase comprises: a) incubating and thereby contacting gOBG3polypeptide fragment with or without a candidate compound and withdipeptidyl peptidase; b) detecting the result, wherein said result iscleavage of the N-terminal dipeptide of OBG3 polypeptide fragment, usinga technology selected from but not restricted to enzyme-linkedimmunosorbent assay (ELISA), mass spectrometry, chromatography,radioisotopic counting, N-terminal sequencing, or functionality whereinsaid functionality is selected from the group consisting of LSRmodulation, leptin modulation, lipoprotein modulation, FFA oxidationmodulation: and c) wherein said result identifies said potentialcompound if said result differs from said result when gOBG3 polypeptidefragment is contacted with dipeptidyl peptidase alone. Preferred saidgOBG3 polypeptide fragment is gOBG3 polypeptide fragment selected fromamino acids 106-247 of SEQ ID NO:2 or SEQ ID NO:4. Most preferred saidgOBG3 polypeptide fragment is gOBG3 polypeptide fragment selected fromamino acids 85-244 or 103-244 of SEQ ID NO:6. Preferred said technologyis ELISA, wherein said ELISA utilizes a monoclonal antibody thatspecifically binds to intact but not to dipeptidyl peptidase cleavedgOBG3 polypeptide fragment. Further preferred said technology is ELISA,wherein said ELISA utilizes a monoclonal antibody that specificallybinds to intact (106-247) but not to dipeptidyl peptidase cleaved(108-247) gOBG3 polypeptide fragment of SEQ ID NO:2 or SEQ ID NO:4. Mostpreferred said technology is ELISA, wherein said ELISA utilizes amonoclonal antibody that specifically binds to intact (85-244) but notto dipeptidyl peptidase cleaved (87-244) gAPM1 polypeptide fragment ofSEQ ID NO:6. Other most preferred said technology is ELISA, wherein saidELISA utilizes a monoclonal antibody that specifically binds to intact(103-244) but not to dipeptidyl peptidase cleaved (105-244) gAPM1polypeptide fragment of SEQ ID NO:6. The invention further featuresmethods for determining the specificity of said antagonist of saiddipeptidyl peptidase cleavage of said gOBG3 polypeptide fragmentcomprising: a) contacting said gOBG3 polypeptide fragment with saidantagonist and said dipeptidyl peptidase; b) contacting an alternativesubstrate of said dipeptidyl peptidase with said antagonist and saiddipeptidyl peptidase; c) detecting a result on the basis of a technologyselected from but not restricted to enzyme-linked immunosorbent assay(ELISA), mass spectrometry, chromatography, radioisotopic counting,N-terminal sequencing, or functionality; and d) wherein said resultidentifies said antagonist as specific for gOBG3 polypeptide fragment ifsaid result differs from said result for said alternative substrate ofsaid dipeptidyl peptidase contacted with said dipeptidyl peptidase andsaid antagonist. Preferred said gOBG3 polypeptide fragment is gACRP30polypeptide fragment of amino acids 106-247 of SEQ ID NO:2 or SEQ IDNO:4; preferred said alternative substrate of said dipeptidyl peptidaseis selected from gAPM1 polypeptide fragment of amino acids 85-244 or103-244 of SEQ ID NO:6. Most preferred said gOBG3 polypeptide fragmentis gAPM1 polypeptide fragment of amino acids 85-244 of SEQ ID NO:6:preferred said alternative substrate of said dipeptidyl peptidase isselected from gAPM1 polypeptide fragment of amino acids 103-244 of SEQID NO: 6 and gOBG3 polypeptide fragment of amino acids 106-247 of SEQ IDNO:2 or SEQ ID NO:4. Other most preferred said gOBG3 polypeptidefragment is gAPM1 polypeptide fragment of amino acids 103-244 of SEQ IDNO:6: preferred said alternative substrate of said dipeptidyl peptidaseis selected from gAPM1 polypeptide fragment of amino acids 85-244 of SEQID NO: 6 and gOBG3 polypeptide fragment of amino acids 106-247 of SEQ IDNO:2 or SEQ ID NO:4.

IX. Assays for Identifying Antagonists of OBG3 or gOBG3 PolypeptideFragment Activity

The invention features methods of screening compounds for one or moreantagonists of OBG3 or gOBG3 polypeptide fragment activity wherein saidactivity is selected from but not restricted to lipid partitioning,lipid metabolism, and insulin-like activity. Preferred said compound isselected from but is not restricted to small molecular weight organic orinorganic compound, protein, peptide, carbohydrate, or lipid. Preferredsaid polypeptide fragment is gOBG3 polypeptide fragment. Preferred saidpolypeptide fragment is gOBG3 polypeptide fragment. Preferred said gOBG3polypeptide fragment is gOBG3 (104-247) of SEQ ID NO:2 or SEQ ID NO:4.Most preferred said gOBG3 polypeptide fragment is gAPM1 (101-244) of SEQID NO:6.

The invention further features methods of screening compounds for saidantagonist of OBG3 or gOBG3 polypeptide fragment activity comprising: a)contacting said gOBG3 polypeptide fragment with or without saidcompound; b) detecting a result on the basis of activity, wherein saidactivity is selected from but not restricted to lipid partitioning,lipid metabolism, and insulin-like activity: and c) wherein said resultidentifies said antagonist as specific for gOBG3 polypeptide fragment ifsaid result with compound differs from said result for said resultwithout compound. Exemplary assays that may be used are described inExamples 4 and 18.

EXAMPLES

The following Examples are provided for illustrative purposes and not asa means of limitation. One of ordinary skill in the art would be able todesign equivalent assays and methods based on the disclosure herein allof which form part of the instant invention.

It should be noted that the term full-length OBG3 polypeptide usedthroughout the specification is intended to encompass the proteinhomologs ACRP30 [Scherer, et al., “A novel serum protein similar to Clq,produced exclusively in adipocytes”; J Biol Chem 270, 26746-26749(1995)], AdipoQ [Hu, et al., “AdipoQ is a novel adipose-specific genedysregulated in obesity”, J Biol Chem 271, 10697-10703 (1996)] and thehuman homolog APM1 [Maeda, et al., “cDNA cloning and expression of anovel adipose specific collagen-like factor, APM1 (AdiPose Most abundantGene transcript 1)”, Biochem Biophys Res Commun 221, 286-289 (1996)] orGBP28 [Nakano, et al., “Isolation and characterization of GBP28, a novelgelatin-binding protein purified from human plasma”, J Biochem (Tokyo)120, 803-812 (1996)]. OBG3 is also intended to encompass other homologs.

Example 1 Production of Recombinant OBG3

An exemplary method for generating recombinant OBG3 is given below.Although the method describes the production of the mouse analog, aperson with skill in the art would be able to use the informationprovided to produce other OBG3 analogs, including but not limited to thehuman analog. An alignment of the amino acid sequences of the human(APM1) and mouse (AdipoQ and ACRP30) OBG3 is shown in FIG. 1.

The recombinant OBG3 analog is cloned in pTRC His B (Invitrogen) betweenBamH1 and Xho1 (FIG. 2) and maintained in E. coli DH5-alpha. Thesequence of the OBG3 insert corresponds to ACRP30 genbank U37222 bases88 to 791 except in position 382 where in #3 G replaces A found in ACRP30 (V instead of M). The corresponding nucleotide in AdipoQ U49915 is Gas in clone #3. The amino acid V is also conserved in the human sequenceAPM1 D45371.

Culture:

Plate out bacteria in LB agar media containing 100 μg/mL ampicillin.Inoculate 1 colony into 5 mL media (no agar) at 37° C., overnight. Add 2mL of this initial culture into 500 mL Erlenmeyer flasks containing 200mL LB media and 100 μg/mL ampicillin. Incubate at 37° C., in an orbitalshaker until the OD₆₀₀=0.2. Add IPTG to a final concentration of 1 mM(stock solution=1 M). Incubate at 37° C., overnight.

Lysis:

Pellet the bacteria by centrifugation (Sorvall, 3500 rpm 15 min, 4° C.)in a pre-weighed tube.

At 4° C., resuspend the pellet in 3 mL/g of lysis buffer

Add 40 μL/g PMSF 10 mM

Add 80 μL/g of lysozyme 10 mg/mL

Incubate 20 min on ice, shaking intermittently

Add 30 μL/g 10% sodium deoxycholate

Incubate at 37° C., until the lysate is viscous

Freeze in liquid Nitrogen and thaw at 37° C., three times

Sonicate 2×, 30 sec, 25% cycle, 2.5 power level

Centrifuge 30 min, 15000 rpm, 4° C.

Recover the supernatant

Note: The lysate can be stored frozen before or after the sonicationstep.

Batch Purification:

1. Pack 1 mL of Probond resin (Invitrogen; 1 mL=2 mL suspended gel) intoa 5 mL column. Wash with 5 mL PBS.

2. Apply 5 mL bacterial supernatant to the 1 mL of gel. (If volume isvery high, use several small columns.)

3. Wash with 24 mL phosphate buffer, pH 7.8, followed by a wash with 24mL phosphate buffer, pH 6.

4. Elute with imidazole buffer and collect fractions of 1 mL.

5. Analyze fractions by OD at 280 nm or by SDS-PAGE (12.5%; dilution ½in 2× sample buffer) under reducing conditions (100° C., 5 min)

6. Pool the fractions containing protein (usually fraction numbers 2-4for concentrations of 0.8-1 mg/mL and fractions 1, 5 and 6 forconcentrations of 0.2-0.4 mg/mL).

7. Dialyze thoroughly against 1×PBS, 24 mM ammonium bicarbonate or 50 mMTris, pH 7.4 containing 250 nM NaCl. Concentrate by Speed-Vac if needed.

8. Analyze protein by the Lowry method.

9. Aliquot and store at −20° C.

Purification on Liquid Chromatography System

1. Pack 5 mL of Probond resin into a 5 mL column.

2. Wash with 4 bed volumes of phosphate buffer pH 7.8, 1 mL/min.

3. Inject 25 mL lysate (filtered on 0.45μ or centrifuged at 3000 rpm, 30min, 4° C., Beckman Allegra 6R) at 0.5 mL/min.

4. Wash with 4 bed volumes of phosphate buffer, pH 7.8 at 1 mL/min.

5. Wash with 12 bed volumes of phosphate buffer pH 5.5 at 1 mL/min.

6. Elute bound fraction with phosphate buffer, pH 5.5, containing 1 Mimidazole at 1 mL/min.

7. Collect fractions, dialyze and analyze protein as described for batchpurification, steps 7-9.

Example 2 Generation of Globular OBG3 by Enzymatic Cleavage

Incubate purified OBG3 (obtained as described above or throughequivalent method) with acetylated Trypsin-Type V-S from Bovine Pancreas(Sigma E.C.=3.4.21.1) at 400 u/mg protein at 25° C., for 10 min.

Stop reaction by running the sample over a Poly-Prep Column (Biorad731-1550) at +4° C. containing immobilized Trypsin inhibitor.

Collect 1.0 mL fractions. Determine protein concentration.

Pool the protein containing fractions and dialyze extensively againstPBS using dialysis tubing with M.W, cutoff=10,000 da.

Concentrate on Amicon YM-10 Centricon Filter (Millipore, M.W,cutoff=10.000 da). Sterile filter.

Determine final protein concentration using Markwell's modified Lowryprocedure (1981) or BCA protein assay (Pierce Chemical Co. Rockford.Ill.) and BSA as standard.

Check purity and efficiency of cleavage by SDS-PAGE analysis using a4-20% gradient gel. The intact OBG3 migrates as a single band atapproximately 37 kDa apparently due to co-transcribed vector sequencesattached to the histidine tag at the N-terminus of AdipoQ, and forms adimer at 74 kDa. The cleaved OBG3 forms a band at approx. 18 kDa(gOBG3). Additional degradation products, all smaller than 10 kda arealso generated from the N-terminal region. These are separated from thedesired 18 kDa band by dialysis with semipermeable membranes with a MWcutoff of 10,000. The two potential cleavage sites for gOBG3 are shownin FIG. 3.

The actual cleavage site has been identified as the one after amino acid103 (amino acid 100 for human gOBG3 or APM1) (FIG. 7). That is, theN-terminus of the gOBG3 cleavage product is Lys 104 (Lys 101 for humangOBG3 or APM1).

Other enzymatic/proteolytic methods can also be used that yield apreferred OBG3 or gOBG3 polypeptide fragment, e.g., clostripain,adipsin, plasmin, collagenase, matrix metalloproteinase-1 (MMP-1), orprecerebellin processing protease. Other preferred enzymes wouldpreferably cleave OBG3 at a site close to the junction between thecollagen-like tail and the globular head (about amino acid 108 for humangOBG3 and about amino acid 11 for murine gOBG3), preferably permit thereaction to be easily stopped, preferably be easily removed using animmobilized inhibitor, or similar method, and preferably cuts theN-terminal fragment into small pieces (less than 10,000 MW). Thecleavage preferably results in the presence of no more than 8 collagenrepeats, more preferably no more than 3 collagen repeats, and mostpreferably no collagen repeats. A collagen repeat consists of GLY-X-Y. Adetermination of whether an active gOBG3 has been generated can bechecked using the in vitro and in vivo assays described herein (Examples4-6, 8-10).

Example 3 Generation of gOBG3 by Recombinant Methodology

Restriction Site Cloning

A first approach is to look for unique restriction sites near thebeginning of the globular head region (nucleic acid sequences of mouseand human OBG3 polypeptides are provided in the sequence listing). Ifpresent, it can be used to cleave within the 5′ collagen-like region andgenerate a C-terminal fragment comprised of the globular head region. Ifa unique site is not present, it is also possible, although moredifficult, to do this using restriction enzymes that cut in more thanone location by doing partial digestions. The 3′ end of the globularhead can be cut from its vector backbone using an appropriate enzyme.The globular head can then be cloned into an expression vector andconstructs containing the correct fragments can be identified. ForAdipoQ. Tau I seems to be a unique enzyme that would separate thecollagen tail from the globular head.

PCR Cloning

Another approach is to PCR the region of interest from the intactsequence (if cDNA is available) using primers with restriction sites onthe end so that PCR products can be directly cloned into vectors ofinterest. Alternatively, gOBG3 can also be generated using RT-PCR toisolate it from adipose tissue RNA.

E. coli Vector

For example, the AdipoQ globular region can be cloned into pTrcHisB, byputting a Bam HI site on the sense oligo and a Xho I site on theantisense oligo. This allows isolation of the PCR product, digestion ofthat product, and ligation into the pTrcHisB vector that has also beendigested with Bam HI and Xho I (FIG. 4). The vector, pTrcHisB, has anN-terminal 6-Histidine tag, that allows purification of the overexpressed protein from the lysate using a Nickel resin column. ThepTrcHisB vector is used for over-expression of proteins in E. coli.

Exemplary oligos for cloning into the E. coli vector include:

A) OBG3 sense CTTAGTGGATCCCGCTTATGTGTATCGCTCAG 6 base pairs from theleft there is a 6 bp BamHI site. Thus the region that is homologous tothe gene begins at nucleotide 13.

B) OBG3 antisense GCTGTTCTCGAGTCAGTTGGTATCATGG 6 base pairs from theleft there is a 6 bp. XhoI site. Thus the region that is homologous tothe gene begins at nucleotide 13.

The following are exemplary PCR conditions.

Final concentrations in the reaction are:

1× PE Biosystems buffer A

1.5 mM MgCl₂

200 uM of each dNTP (dATP, dCTP, dGTP, dTTP)

2.5 Units of Amplitaq Gold from PE Biosystems

0.4 uM of each primer (sense and antisense)

10 ng of plasmid template

Cycling parameters:

95° C. 10 mm—1 cycle

95° C. 30 sec

56° C. 30 sec

72° C. 30 sec

repeat above 3 steps for 30 cycles

72° C. 7 min—1 cycle.

BAC Vector

The globular head can also be over expressed in a Baculovirus systemusing the 6×His Baculovirus kit (Pharmingen), for example. The AdipoQglobular region is cloned into the appropriate vector using enzymesavailable in the multiple cloning site. This allows over-expression ofthe protein in a eukaryotic system which has some advantages over the E.coli system, including: Multiple gene expression, Signal peptidecleavage, Intron splicing, Nuclear transport, Functional protein,Phosphorylation, Glycosylation, and Acylation.

Exemplary oligos for cloning into the Baculovirus vector are thefollowing:

A). OBG3 sense CTTAGTGAATTCGCTTATGTGTATCGCTCAGA 6 base pairs from theleft there is a 6 bp. EcoRI site. Thus the region that is homologous tothe gene begins at nucleotide 13.

B). OBG3 antisense GCTGTTCTGCAGTCAGTTGGTATCATGG 6 base pairs from theleft there is a 6 bp. PstI site. Thus the region that is homologous tothe gene begins at nucleotide 13.

The following are exemplary PCR conditions.

Final concentrations in the reaction are:

1× PE Biosystems buffer A

1.5 mM MgCl,

200 uM of each dNTP (dATP, dCTP, dGTP, dTTP)

2.5 Units of Amplitaq Gold from PE Biosystems

0.4 uM of each primer (sense and antisense)

10 ng of plasmid template

Cycling parameters:

95° C. 10 mm—1 cycle

95° C. 30 sec

60° C. 30 sec

72° C. 30 sec repeat above 3 steps for 30 cycles

72° C. 7 min—1 cycle.

Mammalian Vector

Globular OBG3 can also be cloned into a mammalian expression vector andexpressed in and purified from mammalian cells, for example 3T3-L1 cells(undifferentiated adipocyte precursors). The globular head is thengenerated in an environment very close to its endogenous environment.However, this is not necessarily the most efficient way to make protein.

Example 4 In Vitro Tests of Obesity-related Activity

The activity of various preparations and various sequence variants ofgOBG3 polypeptide fragments are assessed using various in vitro assaysincluding those provided below. These assays are also exemplary of thosethat can be used to develop gOBG3 polypeptide fragment antagonists andagonists. To do that, the effect of gOBG3 polypeptide fragments in theabove assays, e.g., on leptin and/or LSR activity, in the presence ofthe candidate molecules would be compared with the effect of gOBG3polypeptide fragments in the assays in the absence of the candidatemolecules. Since gOBG3 polypeptide fragments have been shown to reducebody weight in mice on a high-cafeteria diet (Example 5), these assaysalso serve to identify candidate treatments for reducing (or increasing)body weight.

Liver Cell Line:

Tests of efficacy of gOBG3 polypeptide fragments on LSR can be performedusing liver cell lines, including for example, PLC. HepG2, Hep3B(human), Hepa 1-6, BPRCL (mouse), or MCA-RH777, MCA-RHS994 (rat). Forhuman cell lines, APM1 and globular APM1 would be used preferentially:for rodents, full-length and globular AdipoQ/ACRP30 would be usedpreferentially.

BPRCL mouse liver cells (ATCC Repository) were plated at a density of300,000 cells/well in 6-well plates (day 0) in DMEM (high glucose)containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992).Media was changed on day 2. On day 3, the confluent monolayers werewashed once with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well).Cells were incubated at 37° C., for 30 min with increasingconcentrations of recombinant AdipoQ (AQ) or globular AdipoQ (AQ-GH) inDMEM containing 0.2% (w/v) BSA, 5 mM Hepes, 2 mM CaCl₂, 3.7 g/L sodiumbicarbonate, pH 7.5. Incubations were continued for 3 h at 37° C., afteraddition of 10 ng/mL ¹²⁵I-mouse leptin (specific activity. 22100cpm/ng). Monolayers were washed 2 times consecutively with PBScontaining 0.2% BSA, followed by 1 wash with PBS/BSA, and then 2 timesconsecutively with PBS. Cells were lysed with 0.1 N NaOH containing 0.24mM EDTA. Lysates were collected into cubes, and counted in agamma-counter. Results of an exemplary experiment are shown as the meanof triplicate determinations in FIG. 5.

The results indicate that gOBG3 polypeptide fragments are at least 30%more efficient than OBG3 in increasing leptin uptake in a liver cellline (FIG. 5). This assay could be used to determine the efficiency ofgOBG3 polypeptide fragments and related compounds (or agonists orantagonists) to increase or decrease leptin uptake into the liver, aswell as the mechanism by which the gOBG3 polypeptide fragment/compoundexerts this effect.

Blood Brain Barrier Model:

The effect of gOBG3 polypeptide fragments on leptin transport in thebrain can be determined using brain-derived cells. One method that isenvisioned is to use the blood/brain barrier model described by Dehouck,et al., “An easier, reproducible, and mass-production method to studythe blood-brain barrier in vitro”, J Neurochem 54, 1798-1801 (1990);hereby incorporated herein by reference in its entirety (including anyfigures, tables, or drawings) that uses a co-culture of brain capillaryendothelial cells and astrocytes to test the effects of gOBG3polypeptide fragments on leptin (or other molecules) transport via LSRor other receptors.

This assay would be an indicator of the potential effect of gOBG3polypeptide fragments on leptin transport to the brain and could be usedto screen gOBG3 polypeptide fragment variants for their ability tomodulate leptin transport through LSR or other receptors in the brain.In addition, putative agonists and antagonists of the effect of gOBG3polypeptide fragments on leptin transport through LSR or other receptorscould also be screened using this assay. Increased transport of leptinacross the blood/brain barrier would presumably increase its action as asatiety factor.

FACS Analysis of LSR Expression

The effect of gOBG3 polypeptide fragments on LSR can also be determinedby measuring the level of LSR expression at the cell surface by flowsurface cytometry, using anti-LSR antibodies and fluorescent secondaryantibodies. Flow cytometry is a laser-based technology that is used tomeasure characteristics of biological particles. The underlyingprinciple of flow cytometry is that light is scattered and fluorescenceis emitted as light from the excitation source strikes the movingparticles.

This is a high through-put assay that could be easily adapted to screenOBG3 and gOBG3 polypeptide fragments and variants as well as putativeagonists or antagonists of gOBG3 polypeptide fragments. Two assays areprovided below. The antibody, cell-line and gOBG3 polypeptide fragmentanalog would vary depending on the experiment, but a human cell-line,human anti-LSR antibody and globular APM1 could be used to screen forvariants, agonists, and antagonists to be used to treat humans.

Assay 1:

Cells are pretreated with either intact OBG3 or gOBG3 polypeptidefragments (or untreated) before harvesting and analysis by FACs. Cellsare harvested using non-enzymatic dissociation solution (Sigma), andthen are incubated for 1 h at 4° C., with a 1:200 dilution of anti-LSR81B or an irrelevant anti-serum in PBS containing 1% (w/v) BSA. Afterwashing twice with the same buffer, goat anti-rabbit FITC-conjugatedantibody (Rockland, Gilbertsville, Pa.) is added to the cells, followedby a further incubation for 30 min at 4° C. After washing, the cells arefixed in 2% formalin. Flow cytometry analysis is done on a FACsCaliburcytometer (Becton-Dickinson, Franklin Lakes, N.J.).

The in vitro Liver Cell Line assay (described above) has shown that LSRactivity (leptin binding) increases with increasing concentrations ofgOBG3 polypeptide fragments. While not wishing to be bound by anyparticular theory, this could either be the result of an increasednumber of LSR binding sites on the cell surface, or a change in affinityfor leptin. The FACs assay would presumably be detecting changes in thenumber of LSR binding sites, although changes in conformation reflectingchanges in affinity might also be detected. Preferably the antibodywould be to the C-terminus of LSR.

Assay 2:

Cells are cultured in T175 flasks according to manufacturer'sinstructions for 48 hours prior to analysis.

Cells are washed once with FACS buffer (1×PBS/2% FBS, filtersterilized), and manually scraped from the flask in 10 mLs of FACSbuffer. The cell suspension is transferred to a 15 mL conical tube andcentrifuged at 1200 rpm, 4° C., for 5 minutes. Supernatant is discardedand cells are resuspended in 10 mL FACS buffer chilled to 4° C. A cellcount is performed and the cell density adjusted with FACS buffer to aconcentration of 1×10⁶ cells/mL. One milliliter of cell suspension wasadded to each well of a 48 well plate for analysis. Cells arecentrifuged at 1200 rpm for 5 minutes at 4° C. Plates are checked toensure that cells are pelleted, the supernatant is removed and cellsresuspended by running plate over a vortex mixer. One milliliter of FACSbuffer is added to each well, followed by centrifugation at 1200 rpm for5 minutes at 4° C. This described cell washing was performed a total of3 times.

Primary antibody, titered in screening experiments to determine properworking dilutions (for example 1:25, 1:50, 1:100, 1:200, 1:400, 1:500,1:800, 1:1000, 1:2000, 1:4000, 1:5000, or 1:10000), is added to cells ina total volume of 50 L FACS buffer. Plates are incubated for 1 h at 4°C., protected from light. Following incubation, cells are washed 3 timesas directed above. Appropriate secondary antibody, titered in screeningexperiments to determine proper working dilutions (for example 1:25,1:50, 1:100, 1:200, 1:400, 1:500, 1:800, 1:1000, 1:2000, 1:4000, 1:5000,or 1:10000), is added to cells in a total volume of 50 μL FACS buffer.Plates are incubated for 1 h at 4° C., protected from light. Followingincubation, cells are washed 3 times as directed above. Upon final wash,cells are resuspended in 500 μL FACS buffer and transferred to a FACSacquisition tube. Samples are placed on ice protected from light andanalyzed within 1 hour.

Cellular Binding and Uptake of gOBG3 as Detected by FluorescenceMicroscopy

Fluorecein isothiocyanate (FITC) conjugation of gOBG3: Purified gOBG3 at1 mg/mL concentration was labeled with FITC using Sigma's FluoroTag FITCconjugation kit (Stock No. FITC-1). Protocol outlined in the SigmaHandbook for small-scale conjugation was followed for gOBG3 labeling.

Cell Culture: C2C12 mouse skeletal muscle cells (ATCC, Manassas, Va.CRL-1772) and Hepa-1-6 mouse hepatocytes (ATCC, Manassas, Va. CRL-1830)were seeded into 6 well plates at a cell density of 2×10⁵ cells perwell. C2C12 and Hepa-1-6 cells were cultured according to repository'sinstructions for 24-48 hours prior to analysis. Assay was performed whencells were 80% confluent.

FITC labeled gOBG3 cellular binding and uptake using microscopy: C2C12and Hepa 1-6 cells were incubated in the presence/absence of antibodydirected against human LSR (81B: N-terminal sequence of human LSR; doesnot cross react with mouse LSR and 93A: c-terminal sequence, crossreacts with mouse LSR) or an antiserum directed against gC1qr (953) for1 hour at 37° C., 5% CO₂. LSR antibodies were added to the media at aconcentration of 2 μg/mL. The anti-gC1qr antiserum was added to themedia at a volume of 2.5 μL undiluted serum (high concentration) or1:100 dilution (low concentration). Following incubation with specifiedantibody, FITC-gOBG3 (50 nM/mL) was added to each cell culture well.Cells were again incubated for 1 hour at 37° C., 5% CO2. Cells werewashed 2× with PBS, cells were scraped from well into 1 mL of PBS. Cellsuspension was transferred to an eppendorf tube and centrifuged at 1000rpm for 2 minutes. Supernatant was removed and cells resuspended in200.μL of PBS. Binding and uptake of FITC-gOBG3 was analyzed byfluorescence microscopy under 40× magnification.

Analysis of C2C12 and Hepa 1-6 cells reveals identical phenotypes withrespect to FITC-gOBG3 binding and uptake profiles both in the presenceor absence of LSR antibodies. FITC-gOBG3 appears to be localized withinvesicles in the cytoplasm of both mouse hepatocytes and mouse myoblasts,suggesting that binding and uptake of FITC-gOBG3 is occurring.FITC-gOBG3 uptake appears to be blocked when cells were pre-treated withthe anti-LSR antibody that recognizes mouse LSR. However, binding ofFITC-gOBG3 to the cell surface does occur in a small portion of thecells (C2C12 and Hepa 1-6). At low concentration of the gClqr antiserum,FITC-gOBG3 appears to be localized within vesicles in the cytoplasm ofboth cell types, similarly to the phenotype of cells that have notreceived antibody pre-treatment prior to addition of FITC-gOBG3.FITC-gOBG3 uptake and binding phenotype is not affected by pre-treatmentwith an LSR antibody that does not recognize mouse LSR. Together, thesedata suggest that uptake of FITC-gOBG3 can be blocked by a human LSRantibody which cross-reacts with mouse LSR. However, this phenotype isnot reproduced with other non cross-reactive LSR antibodies. Thus, thisassay may be useful for identifying agents that facilitate or preventthe uptake and/or binding of OBG3 or gOBG3 polypeptide fragments tocells.

Effect on LSR as a Lipoprotein Receptor

The effect of gOBG3 on the lipoprotein binding, internalizing anddegrading activity of LSR can also be tested. Measurement of LSR aslipoprotein receptor is described in Bihain & Yen, ((1992) Biochemistry31(19):4628-36; hereby incorporated herein in its entirety including anydrawings, tables, or figures). The effect of gOBG3 on the lipoproteinbinding, internalizing and degrading activity of LSR (or otherreceptors) can be compared with that of intact OBG3, with untreatedcells as an additional control. This assay can also be used to screenfor active and inhibitory variants of gOBG3, as well as agonists andantagonists of obesity-related activity.

Human liver PLC cells (ATCC Repository) were plated at a density of300,000 cells/well in 6-well plates (day 0) in DMEM (high glucose)containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992).Media was changed on day 2. On day 3, the confluent monolayers werewashed once with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well).Cells were incubated at 37° C., for 30 min with 10 ng/mL humanrecombinant leptin in DMEM containing 0.2% (w/v) BSA, 5 mM Hepes, 2 mMCaCl, 3.7 g/L sodium bicarbonate, pH 7.5, followed by another 30 minincubation at 37° C., with increasing concentrations of gOBG3.Incubations were continued for 2 h at 37° C., after addition of 0.8 mMoleate and 20 μg/mL ¹²⁵ I-LDL. Monolayers were washed 2 timesconsecutively with PBS containing 0.2% BSA, followed by 1 wash withPBS/BSA, and then 2 times consecutively with PBS. The amounts ofoleate-induced binding, uptake and degradation of ¹²⁵I-LDL were measuredas previously described (Bihain & Yen. 1992, supra). Results are shownas the mean of triplicate determinations.

As shown in FIG. 6, the addition of gOBG3 leads to an increased activityof LSR as a lipoprotein receptor. The oleate-induced binding and uptakeof LDL appears more affected by gOBG3 as compared to the degradation.This increased LSR activity would potentially result in an enhancedclearance of triglyceride-rich lipoproteins during the postprandialstate. Thus, more dietary fat would be removed through the liver, ratherthan being deposited in the adipose tissue. This assay could be used todetermine the efficiency of a compound (or agonists or antagonists) toincrease or decrease LSR activity (or lipoprotein uptake, binding anddegradation through other receptors), and thus affect the rate ofclearance of triglyceride-rich lipoproteins.

Effect on Muscle Differentiation

C2C12 cells (murine skeletal muscle cell line; ATCC CRL 1772, Rockville,Md.) are seeded sparsely (about 15-20%) in complete DMEM (w/glutamine,pen/strep, etc)+10% FCS.

Two days later they become 80-90% confluent. At this time, the media ischanged to DMEM+2% horse serum to allow differentiation. The media ischanged daily. Abundant myotube formation occurs after 3-4 days of beingin 2% horse serum, although the exact time course of C2C12differentiation depends on how long they have been passaged and how theyhave been maintained, among other things.

To test the effect of the presence of gACRP30 on muscle differentiation,gACRP30 (1 to 2.5 μg/mL) was added the day after seeding when the cellswere still in DMEM w/10% FCS. Two days after plating the cells (one dayafter gACRP30 was first added), at about 80-90% confluency, the mediawas changed to DMEM+2% horse serum plus gACRP30. The results show thatthe addition of gACRP30 causes the cells to begin organizing within oneday after its addition. In contrast to the random orientation of thecells not treated with gACRP30, those treated with gACRP30 alignedthemselves in relation to each other. In addition, differentiationoccurred after only 2 days of gACRP30 treatment, in contrast to the 3 to4 days needed in its absence.

Effect on Muscle Cell Fatty Acid Oxidation

C2C12 cells were differentiated in the presence or absence of 2 μg/mLgACRP30 for 4 days. On day 4, oleate oxidation rates were determined bymeasuring conversion of 1-¹⁴C-oleate (0.2 mM) to ¹⁴CO₂ for 90 min. C2C12cells differentiated in the presence of gACRP30 undergo 40% more oleateoxidation than controls differentiated in the absence of gACRP30. Thisexperiment can be used to screen for active fragments and peptides aswell as agonists and antagonists or activators and inhibitors of OBG3and gOBG3 polypeptides.

The effect of gACRP30 on the rate of oleate oxidation was compared indifferentiated C2C12 cells (murine skeletal muscle cells; ATCC.Manassas, Va. CRL-1772) and in a hepatocyte cell line (Hepal-6: ATCC.Manassas. VA CRL-1830). Cultured cells were maintained according tomanufacturer's instructions. The oleate oxidation assay was performed aspreviously described (Muoio et al (1999) Biochem J 338:7 S3-791).Briefly, nearly confluent myocytes were kept in low serumdifferentiation media (DMEM, 2.5% Horse serum) for 4 days, at which timeformation of myotubes became maximal. Hepatocytes were kept in the sameDMEM medium supplemented with 10% FCS for 2 days. One hour prior to theexperiment the media was removed and 1 mL of preincubation media (MEM,2.5% Horse serum, 3 mM glucose, 4 mM Glutamine, 25 mM Hepes. 1% FFA freeBSA, 0.25 mM Oleate, 5 μg/mL gentamycin) was added. At the start of theoxidation experiment ¹⁴C-Oleic acid (1 μCi/mL. American RadiolabeledChemical Inc. St. Louis. Mo.) was added and cells were incubated for 90min at 37° C., in the absence/presence of 2.5 μg/mL gACRP30. After theincubation period 0.75 mL of the media was removed and assayed for¹⁴C-oxidation products as described below for the muscle FFA oxidationexperiment.

Oleate oxidation in C2C12 cells determined over 90 min increasedsignificantly (39%; p=0.036, two-tailed t-Test) in cells treated withgACRP30. In contrast, no detectable increase in the rate of FFAoxidation was seen in hepatocytes incubated with gACRP30.

Triglyceride and Protein Analysis Following Oleate Oxidation in CulturedCells

Following transfer of media for oleate oxidation assay, cells wereplaced on ice. To determine triglyceride and protein content, cells werewashed with 1 mL of 1×PBS to remove residual media. To each well 300 μLof cell dissociation solution (Sigma) was added and incubated at 37° C.,for 10 min. Plates were tapped to loosen cells, and 0.5 mL of 1×PBS wasadded. The cell suspension was transferred to an eppendorf tube, eachwell was rinsed with an additional 0.5 mL of 1×PBS, and was transferredto appropriate eppendorf tube. Samples were centrifuged at 1000 rpm for10 minutes at room temperature. Supernatant was discarded and 750 μL of1×PBS/2% chaps was added to cell pellet. Cell suspension was vortexedand placed on ice for 1 hour. Samples were then centrifuged at 13000 rpmfor 20 min at 4° C. Supernatants were transferred to new tube and frozenat −20° C., until analyzed. Quantitative measure of triglyceride levelin each sample was determined using Sigma Diagnostics GPO-TRINDERenzymatic kit. The procedure outlined in the manual was adhered to, withthe following exceptions: assay was performed in 48 well plate, 350 L ofsample volume was assayed, control blank consisted of 350 μL PBS/2%chaps, and standard contained 10 μL standard provided in kit plus 690 LPBS/2% chaps. Analysis of samples was carried out on a Packard SpectraCount at a wavelength of 550 nm. Protein analysis was carried out on 25μL of each supernatant sample using the BCA protein assay (Pierce)following manufacturer's instructions. Analysis of samples was carriedout on a Packard Spectra Count at a wavelength of 550 nm.

Triglyceride production in both C2C12 and Hepa 1-6 cells did not changesignificantly in the absence/presence of ACRP30 and gACRP30. The proteincontent of all cells analyzed was equivalent in the absence/presence ofACRP30 and gACRP30.

In Vitro Glucose Uptake by Muscle Cells

L6 Muscle cells are obtained from the European Culture Collection(Porton Down) and are used at passages 7-11. Cells are maintained instandard tissue culture medium DMEM, and glucose uptake is assessedusing [³H]-2-deoxyglucose (2DG) with or without OBG3 or gOBG3polypeptide fragment in the presence or absence of insulin (10⁻⁸ M) ashas been previously described (Walker, P. S, et al. (1990) Glucosetransport activity in L6 muscle cells is regulated by the coordinatecontrol of subcellular glucose transporter distribution, biosynthesis,and mRNA transcription. JBC 265(3):1516-1523; and Kilp, A, et al. (1992)Stimulation of hexose transport by metformin in L6 muscle cells inculture. Endocrinology 130(5):2535-2544, which disclosures are herebyincorporated by reference in their entireties). Uptake of 2DG isexpressed as the percentage change compared with control (no addedinsulin or OBG3 or gOBG3 polypeptide fragment). Values are presented asmean±SEM of sets of 4 wells per experiment.

Differences

between sets of wells are evaluated by Student's t test, probabilityvalues p<0.05 are considered to be significant,

Example 5 Effect of gOBG3 on Mice Fed a High-Fat Diet

Experiments are performed using approximately 6 week old C57Bl/6 mice (8per group). All mice are housed individually. The mice are maintained ona high fat diet throughout each experiment. The high fat diet (cafeteriadiet; D12331 from Research Diets., Inc.) has the following composition:protein kcal % 16, sucrose kcal % 26, and fat kcal % 58. The fat wasprimarily composed of coconut oil, hydrogenated.

After the mice are fed a high fat diet for 6 days, micro-osmotic pumpsare inserted using isoflurane anesthesia, and are used to provide gOBG3,OBG3, saline, and an irrelevant peptide to the mice subcutaneously(s.c.) for 18 days, gOBG3 is provided at doses of 50, 25, and 2.5μg/day: OBG3 is provided at 100, 50, and 5 μg/day; and the irrelevantpeptide is provided at 10 μg/day. Body weight is measured on the first,third and fifth day of the high fat diet, and then daily after the startof treatment. Final blood samples are taken by cardiac puncture and areused to determine triglyceride (TG), total cholesterol (TC), glucose,leptin, and insulin levels. The amount of food consumed per day is alsodetermined for each group.

In a preliminary experiment, mice treated with 2.5 μg/day gOBG3 hadsignificantly lowered body weight.

Example 6 Tests of Obesity-related Activity in Humans

Tests of the efficacy of gOBG3 in humans are performed in accordancewith a physicians recommendations and with established guidelines. Theparameters tested in mice are also tested in humans (e.g., food intake,weight. TG. TC, glucose, insulin, leptin. FFA). It is expected that thephysiological factors would show changes over the short term. Changes inweight gain might require a longer period of time. In addition, the dietwould need to be carefully monitored. Globular OBG3 would be given indaily doses of about 6 mg protein per 70 kg person or about 10 mg perday. Other doses would also be tested, for instance 1 mg or 5 mg per dayup to 20 mg, 50 mg, or 100 mg per day.

Example 7 In Vivo Tests for Obesity-Related Activity in Rodent DiabetesModels

As metabolic profiles differ among various animal models of obesity anddiabetes, analysis of multiple models is undertaken to separate theeffects of OBG3 or gOBG3 polypeptide fragment on hyperglycemia,hyperinsulinemia, hyperlipidemia, and obesity. Mutation in colonies oflaboratory animals and different sensitivities to dietary regimens havemade the development of animal models with non-insulin dependentdiabetes associated with obesity and insulin resistance possible.Genetic models such as db/db and ob/ob (See Diabetes (1982) 31(1):1-6)in mice and fa/fa in zucker rats have been developed by the variouslaboratories for understanding the pathophysiology of disease and fortesting the efficacy of new antidiabetic compounds (Diabetes (1983) 32:830-838; Annu Rep Sankyo Res Lab (1994) 46:1-57). Homozygous C57BL/KsJ-db/db mice developed by Jackson Laboratory are obese,hyperglycemic, hyperinsulinemic, and insulin resistant (J Clin Invest(1990) 85:962-967), whereas heterozygous mice are lean andnormoglycemic. In db/db model, mice progressively develop insulinopeniawith age, a feature commonly observed in late stages of human type IIdiabetes when blood sugar levels are insufficiently controlled. Thestate of the pancreas and its course vary according to the models. Sincethis model resembles that of type II diabetes mellitus, the compounds ofthe present invention are tested for blood sugar and triglycerideslowering activities. Zucker (fa/fa) rats are severely obese,hyperinsulinemic, and insulin resistant (Coleman. Diabetes 31:1, 1982;E. Shafrir, in Diabetes Mellitus: H. Rifkin and D. Porte, Jr. Eds.(Elsevier Science Publishing Co., Inc., New York, ed. 4, 1990), pp.299-340), and the fa/fa mutation may be the rat equivalent of the murinedb mutation (Friedman et al. Cell 69:217-220, 1992: Truett et al., ProcNatl Acad Sci USA 88:7806, 1991). Tubby (tub/tub) mice are characterizedby obesity, moderate insulin resistance and hyperinsulinemia withoutsignificant hyperglycemia (Coleman et al., J Heredit 81:424, 1990).

Previously, leptin was reported to reverse insulin resistance anddiabetes mellitus in mice with congenital lipodystrophy (Shimomura etal. Nature 401:73-76 (1999): hereby incorporated herein in its entiretyincluding any drawings, figures, or tables). Leptin was found to be lesseffective in a different lipodystrophic mouse model of lipoarrophicdiabetes (Gavrilova et al Nature 403:850 (2000): hereby incorporatedherein in its entirety including any drawings, figures, or tables). Theinstant invention encompasses the use of OBG3 or gOBG3 polypeptidefragments for reducing the insulin resistance and hyperglycaemia in thismodel either alone or in combination with leptin, the leptin peptide(U.S. Provisional Application No. 60/155,506), or other compounds.Assays include that described previously in Gavrilova et al. ((2000)Diabetes 49(11):1910-6: (2000) Nature 403(6772):850) using A-ZIP,F-1mice, except that OBG3 or gOBG3 polypeptide fragment would beadministered using the methods previously described in Example 5 (orExamples 8-10). The glucose and insulin levels of the mice would betested, and the food intake and liver weight monitored, as well as otherfactors, such as leptin, FFA, and TG levels, typically measured in ourexperiments (see Example 5, above, or Examples 8-10).

The streptozotocin (STZ) model for chemically-induced diabetes is testedto examine the effects of hvperglycemia in the absence of obesity.STZ-treated animals are deficient in insulin and severely hyperglycemic(Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H.Rifkin and D. Porte. Jr. Eds. (Elsevier Science Publishing Co., Inc.,New York, ed. 4, 1990), pp. 299-340). The monosodium glutamate (MSG)model for chemically-induced obesity (Olney, Science 164:719, 1969;Cameron et al., Clin Exp Pharmacol Physiol 5:41, 1978), in which obesityis less severe than in the genetic models and develops withouthyperphagia, hyperinsulinemia and insulin resistance, is also examined.Finally, a non-chemical, non-genetic model for induction of obesityincludes feeding rodents a high fat/high carbohydrate (cafeteria diet)diet ad libitum.

The instant invention encompasses the use of OBG3 and gOBG3 polypeptidefragment for reducing the insulin resistance and hyperglycemia in any orall of the above rodent diabetes models or in humans with Type I or TypeII diabetes or other preferred metabolic diseases described previouslyor models based on other mammals. In the compositions of the presentinvention the OBG3 or gOBG3 polypeptide fragment may, if desired, beassociated with other compatible pharmacologically-active antidiabeticagents such as insulin, leptin (U.S. Provisional Application No.60/155,506), or troglitazone, either alone or in combination. Assaysinclude that described previously in Gavrilova et al. ((2000) Diabetes49(11):1910-6: (2000) Nature 403(6772):850) using A-ZIP/F-1 mice, exceptthat OBG3 or gOBG3 polypeptide fragment is administeredintraperitoneally (i.p.), subcutaneously (s.c.), intramuscularly (i.m.),or intravenously (i.v.). The glucose and insulin levels of the micewould be tested, and the food Intake and liver weight monitored, as wellas other factors, such as leptin. FFA, and TG levels, typically measuredin our experiments.

In Vivo Assay for Anti-Hyperglycemic Activity of OBG3 or gOBG3Polypeptide Fragment

Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old)are housed (7-9 mice/cage) under standard laboratory conditions at 22°C., and 50% relative humidity, and maintained on a diet of Purina rodentchow and water ad libitum. Prior to treatment, blood is collected fromthe tail vein of each animal and blood glucose concentrations aredetermined using One Touch Basic Glucose Monitor System (Lifescan). Micethat have plasma glucose levels between 250 to 500 mg/dl are used. Eachtreatment group consists of seven mice that are distributed so that themean glucose levels are equivalent in each group at the start of thestudy, db/db mice are dosed by micro-osmotic pumps, inserted usingisoflurane anesthesia, to provide OBG3 or gOBG3 polypeptide fragment,saline, and an irrelevant peptide to the mice subcutaneously (s.c.).Blood is sampled from the tail vein hourly for 4 hours and at 24, 30 hpost-dosing and analyzed for blood glucose concentrations. Food iswithdrawn from 0-4 h post dosing and reintroduced thereafter. Individualbody weights and mean food consumption (each cage) are also measuredafter 24 h. Significant differences between groups (comparing OBG3 orgOBG3 fragment treated to saline-treated) are evaluated using Studentt-test.

In Vivo Insulin Sensitivity Assay

In vivo insulin sensitivity is examined by utilizing two-stephyperinsulinemic-euglycemic clamps according to the following protocol.Rodents from any or all of the various models described in Example 2 arehoused for at least a week prior to experimental procedures. Surgeriesfor the placement of jugular vein and carotid artery catheters areperformed under sterile conditions using ketamine and xylazine (i.m.)anesthesia. After surgery, all rodents are allowed to regainconsciousness and placed in individual cages. OBG3 or gOBG3 polypeptidefragment or vehicle is administered through the jugular vein aftercomplete recovery and for the following two days. Sixteen hours afterthe last treatment, hyperinsulinemic-euglycemic clamps are performed.Rodents are placed in restrainers and a bolus of 4 μCi [3-³H] glucose(NEN) is administered, followed by a continuous infusion of the tracerat a dose of 0.2 μCi/min (20 μl/min). Two hours after the start of thetracer infusion. 3 blood samples (0.3 ml each) are collected at 10minute intervals (−20-0 min) for basal measurements. An insulin infusionis then started (5 mU/kg/min), and 100 μl blood samples are taken every10 min, to monitor plasma glucose. A 30% glucose solution is infusedusing a second pump based on the plasma glucose levels in order to reachand maintain euglycemia. Once a steady state is established at 5mU/kg/min insulin (stable glucose infusion rate and plasma glucose). 3additional blood samples (0.3 ml each) are obtained for measurements ofglucose. [3-³H] glucose and insulin (100-120 min.). A higher dose ofinsulin (25 mU/kg/min.) is then administered and glucose infusion ratesare adjusted for the second euglycemic clamp and blood samples are takenat 220-240 min. Glucose specific activity is determined in deproteinizedplasma and the calculations of Rd and hepatic glucose output (HGO) aremade, as described (Lang et al., Endocrinology 130:43, 1992). Plasmainsulin levels at basal period and after 5 and 25 mU/kg/min, infusionsare then determined and compared between OBG3 or gOBG3 fragment treatedand vehicle treated rodents. Insulin regulation of glucose homeostasishas two major components; stimulation of peripheral glucose uptake andsuppression of hepatic glucose output. Using tracer studies in theglucose clamps, it is possible to determine which portion of the insulinresponse is affected by GBG3 or gOBG3 polypeptide fragment.

Example 8 Effect of gOBG-3 on Plasma Free Fatty Acid in C57 BL/6 Mice

The effect of the globular head of ACRP30 on postprandial lipemia (PPL)in normal C57BL6/J mice was tested. ACRP30 is another name for adipo Qand is the mouse protein homologue to the human APM1 protein. OBG3 is ageneric way to refer to all of these forms. The globular head form isindicated by placing a ‘g’ in front, e.g., gACRP30 or gOBG3. The gOBG3used was prepared by proteolytic digestion of recombinant OBG3 asdescribed previously in Example 2. Acetylated trypsin was used asprotease.

The mice used in this experiment were fasted for 2 hours prior to theexperiment after which a baseline blood sample was taken. All bloodsamples were taken from the tail using EDTA coated capillary tubes (50μL each time point). At time 0 (8:30 AM), a standard high fat meal (6 gbutter, 6 g sunflower oil, 10 g nonfat dry milk, 10 g sucrose, 12 mLdistilled water prepared fresh following Nb#6. IF, pg. 1) was given bygavage (vol.=1% of body weight) to all animals.

Immediately following the high fat meal, 25 μg gOBG3 was injected i.p,in 100 μL saline.

The same dose (25 μg/mL in 100 μL) was again injected at 45 min and at 1hr 45 min (treated croup, n=8). Control animals (n=8) were injected withsaline (3×100 μL). Untreated and treated animals were handled in analternating mode.

Blood samples were taken in hourly intervals, and were immediately puton ice. Plasma was prepared by centrifugation following each time point.Plasma was kept at −20° C., and free fatty acids (FFA), triglycerides(TG) and glucose were determined within 24 hours using standard testkits (Sigma and Wako). Due to the limited amount of plasma available,glucose was determined in duplicate using pooled samples. For each timepoint, equal volumes of plasma from all 8 animals per treatment groupwere pooled. Error bars shown for glucose therefore represent the SD ofthe duplicate determination and not the variation between animals as forTG and FFA.

Results

The increase in plasma FFA due to the high fat meal was significantlylower in mice treated with gOBG3 at all time points between 1 and 4 hr.This can be interpreted as increase in FFA oxidation (FIG. 8).

Treatment with gOBG3 also led to a significantly smaller increase inplasma TG compared to untreated mice. However, this effect was lesspronounced than the effect on FFA (FIG. 9).

Glucose turnover was significantly improved following treatment withgOBG3 this effect can be interpreted as improved insulin sensitivitypossibly due to the decrease in FFA (FIG. 10).

Similar results were seen previously in a prior experiment involvingonly 2 treatments (at 0 and at 45 minutes; data not shown). A strong FFAlowering effect of gOBG3 coupled with a less dominant TG lowering effectwas observed.

Example 9 Effect of gOBG3 on Plasma Leptin and Insulin in C57 BL/6 Mice

The effect of the globular head of ACRP30 on plasma leptin and insulinlevels during postprandial lipemia (PPL) in normal C57BL6/J mice wastested. The experimental procedure was the same as that described inExample 8, except that blood was drawn only at 0, 2 and 4 hours to allowfor greater blood samples needed for the determination of leptin andinsulin by RIA.

Briefly, 16 mice were fasted for 2 hours prior to the experiment afterwhich a baseline blood sample was taken. All blood samples were takenfrom the tail using EDTA coated capilliary tubes (100 μL each timepoint). At time 0 (9:00 AM), a standard high fat meal (see Example 8)was given by gavage (vol.=1% of body weight) to all animals. Immediatelyfollowing the high fat meal, 25 μg gOBG3 was injected i.p, in 100 μLsaline. The same dose (25 g in 100 μL) was again injected at 45 min andat 1 hr 45 min (treated group, n=8). Control animals (n=8) were injectedwith saline (3×100 μL). Untreated and treated animals were handled in analternating mode.

Blood samples were immediately put on ice and plasma was prepared bycentrifugation following each time point. Plasma was kept at −20° C.,and free fatty acids (FFA) were determined within 24 hours using astandard test kit (Wako). Leptin and Insulin were determined by RIA(ML-82K and SRI-13K. LINCO Research, Inc., St. Charles. Mo.) followingthe manufacturer's protocol. However, only 20 μL plasma was used. Eachdetermination was done in duplicate. Due to the limited amount of plasmaavailable, leptin and insulin were determined in 4 pools of 2 animalseach in both treatment groups.

Results

As shown previously (Example 8), treatment with OBG3 significantlyreduced the postprandial increase in plasma FFA caused by the high fatmeal at 2 hours (FIG. 11). There was no significant change in plasmaleptin levels at any time point: treatment with gOBG3 did not affectleptin levels (FIG. 12). Insulin levels (FIG. 13) indicate a marginalincrease in insulin at 2 hours. However, when analyzed as percentagechange from to, this increase (212% vs. 260%, control vs. treated) wasstatistically not significant (p=0.09).

These data reconfirm the previously shown acceleration of FFA metabolismby treatment with gOBG3. They also show that gOBG3 does not affectleptin and insulin plasma levels and that gOBG3 reduces hyperglycemiaduring postprandial lipemia and also induces weight loss duringtreatment over several days. Without being limited by any particulartheory, the data suggests: a) that the reduction in weight is caused bya leptin independent increase in metabolism; and b) that gOBG3 leads toincreased insulin sensitivity.

Example 10 Effect of OBG3 on Plasma FFA. TG and Glucose in C57 BL/6 Mice

The effect of the globular head of ACRP30 on plasma FFA, TG, glucose,leptin and insulin levels during postprandial lipemia (PPL) in normalC57BL6/J mice has been described. Weight loss resulting from gOBG3 (2.5μg/day) given to normal C57BL6/J mice on a high fat diet has also beenshown (Example 5). In comparison, a much higher dose of the completeform of ACRP30 (200 μg/day) was needed to induce a relatively smallereffect in mice. This example shows the effect of the ACRP30-completeform on plasma FFA, TG and glucose levels.

The experimental procedure was similar to that described in Example 8.Briefly, 14 mice were fasted for 2 hours prior to the experiment afterwhich a baseline blood sample was taken. All blood samples were takenfrom the tail using EDTA coated capillary tubes (50 μL each time point).At time 0 (9:00 AM), a standard high fat meal (see Example 8) was givenby gavage (vol.=1% of body weight) to all animals. Immediately followingthe high fat meal, 4 mice were injected 25 μg OBG3 i.p, in 100 μLsaline. The same dose (25 μg in 100 μL) was again injected at 45 min andat 1 hr 45 min. A second treatment group (n=4) received 3 times 50 μgOBG3 at the same intervals. Control animals (n=6) were injected withsaline (3×100 μL). Untreated and treated animals were handled in analternating mode.

Blood samples were immediately put on ice. Plasma was prepared bycentrifugation following each time point. Plasma was kept at −20° C.,and free fatty acids (FFA), triglycerides (TG) and glucose weredetermined within 24 hours using standard test kits (Sigma and Wako).

Results

Treatment with full-length OBG3 had no effect on plasma FFA levels (FIG.14) except for t=2 hours when a statistically significant reduction wasshown (p<0.05). No significant change in postprandial TG (FIG. 15) andglucose levels (FIG. 16) was seen in treated animals.

The data presented show that the complete form of OBG3 did not reduceFFA. TG and glucose levels in contrast to what was observed for theglobular region (Examples 5, 8, 9). Only at 2 hours post-gavage, didtreatment with OBG3 reduce FFA plasma concentrations significantly(p<D.05). These results demonstrate that gOBG3 is much more active invivo than the full-length protein. A similar effect was seen for bodyweight reduction: the globular head was much more active than thefull-length protein.

Example 11 Effect of gACRP30 on FFA Following Epinephrine Injection

In mice, plasma free fatty acids increase after intragastricadministration of a high fat/sucrose test meal. These free fatty acidsare mostly produced by the activity of lipolytic enzymes i.e.,lipoprotein lipase (LPL) and hepatic lipase (HL). In this species, theseenzymes are found in significant amounts both bound to endothelium andfreely circulating in plasma. Another source of plasma free fatty acidsis hormone sensitive lipase (HSL) that releases free fatty acids fromadipose tissue after β-adrenergic stimulation. To test whether gACRP30also regulates the metabolism of free fatty acid released by HSL, micewere injected with epinephrine.

Two groups of mice (n=5 each) were given epinephrine (5 kg) byintraperitoneal injection. A treated group was injected with gACRP30 (25μg) one hour before and again together with epinephrine, while controlanimals received saline. Plasma was isolated and free fatty acids andglucose were measured as described above (Example 10). As shown in FIG.18, epinephrine injections (5 μg) caused an increase in plasma freefatty acids and glucose. Both effects were significantly reduced in2ACRP30− treated mice.

This reduction in the increases of glucose and FFA levels was not due toblockage of the β-adrenergic effect of epinepmephrine, as shown byinducing the release of FFA from isolated adipose tissue in vitro. Inthese control studies, adipose tissue was removed from normal C57BL/6Jmice and incubated in Krebs-Henseleit bicarbonate buffer. Epinephrinewas added and the concentration of FFA in the medium following a 90 minincubation was determined. Epinephrine (10 μM) caused a 1.7-foldincrease in free fatty acids in the media. Increasing concentrations ofgACRP30 or ACRP30 up to 50 μg/ml did not inhibit this effect ofepinephrine. The data presented thus far indicate that the globularregion of ACRP30 exerts profound pharmacological effects on themetabolism of energy substrates with the most evident effect on plasmafree fatty acids. Further, the reduction in plasma FFA concentrationcannot be explained by inhibition of either LPL which would cause anincrease in plasma triglycerides while a decrease of plasmatriglycerides is actually observed, or by inhibition of HSL. Thus, thesimplest explanation is that gACRP30 causes increased removal of freefatty acids from the circulation by promoting cellular uptake.

Example 12 Effect of gACRP30 on Muscle FFA Oxidation

To investigate the effect of gACRP30 on muscle free fatty acidoxidation, intact hind limb muscles from C57BL/6J mice were isolated andFFA oxidation was measured using oleate as substrate [Clee, et al.,“Plasma and vessel wall lipoprotein lipase have different roles inaherosclerosis”, J Lipid Res 41, 521-531 (2000): Muoio, et al. “Leptinopposes insulin's effects on far acid partitioning in muscles isolatedfrom obese ob/ob mice”, Am J Physiol 276, E913-921 (1999). Oleateoxidation in isolated muscle was measured as previously described[Cuendet, et al., “Decreased basal, noninsulin-stimulated glucose uptakeand metabolism by skeletal soleus muscle isolated fromobese-hyperglycemic (ob/ob) mice”, J Clin Invest 58, 1078-1088 (1976);Le Marchand-Brustel, et al., “Insulin binding and effects in isolatedsoleus muscle of lean and obese mice”, Am J Physiol 234, E348-E35S(1978)]. Briefly, mice were sacrificed by cervical dislocation andsoleus and EDL muscles were rapidly isolated from the hind limbs. Thedistal tendon of each muscle was tied to a piece of suture to facilitatetransfer among different media. All incubations were carried out at 30°C., in 1.5 mL of Krebs-Henseleit bicarbonate buffer (118.6 mM NaCl, 4.76mM KCl. 1.19 mM KH₂PO₄, 1.19 mM MgSO₄, 2.54 mM CaCl₂, 25 mM NaHCO₃, 10mM Hepes, pH 7.4) supplemented with 4% FFA free bovine serum albumin(fraction V, RIA grade. Sigma) and 5 mM glucose (Sigma). The totalconcentration of oleate (Sigma) throughout the experiment was 0.25 mM.All media were oxygenated (95% O₂; 5% CO₂) prior to incubation. The gasmixture was hydrated throughout the experiment by bubbling through a gaswasher (Kontes Inc., Vineland. NJ).

Muscles were rinsed for 30 min in incubation media with oxygenation. Themuscles were then transferred to fresh media (1.5 mL) and incubated at30° C., in the presence of 1 μCi/mL [1-¹⁴C]oleic acid (AmericanRadiolabeled Chemicals). The incubation vials containing this media weresealed with a rubber septum from which a center well carrying a piece ofWhatman paper (1.5 cm×11.5 cm) was suspended.

After an initial incubation period of 10 min with constant oxygenation,gas circulation 1 was removed to close the system to the outsideenvironment and the muscles were incubated for 90 min at 30° C. At theend of this period, 0.45 mL of Solvable (Packard Instruments. Meriden,Conn.) was injected onto the Whatman paper in the center well and oleateoxidation by the muscle was stopped by transferring the vial onto ice.

After 5 min, the muscle was removed from the medium, and an aliquot of0.5 mL medium was also removed. The vials were closed again and 1 mL of35% perchloric acid was injected with a syringe into the media bypiercing through the rubber septum. The CO₂ released from the acidifiedmedia was collected by the Solvable in the center well. After a 90 mincollection period at 30° C., the Whatman paper was removed from thecenter well and placed in scintillation vials containing 15 mL ofscintillation fluid (HionicFlour, Packard Instruments, Meriden, Conn.).The amount of ¹⁴C radioactivity was quantitated by liquid scintillationcounting. The rate of oleate oxidation was expressed as nmol oleateproduced in 90 min/g muscle.

To test the effect of aACRP30 or ACRP30 on oleate oxidation, theseproteins were added to the media at a final concentration of 2.5 Lug/mLand maintained in the media throughout the procedure.

Two muscles of different oxidative capacity (soleus and extensordigitorum longus (EDL)) were tested (FIG. 19). EDL and Soleus muscleswere isolated from both legs of normal C57BL/6J mice (n=1S). One muscleof each pair was incubated in medium with 2.5 μg/mL gACRP30 (dark gray)and one in medium without gACRP30 (control-light gray). Thisexperimental design allowed us to compare oleate oxidation in pairs ofmuscles isolated from the same animal. ¹⁴C-Oleate oxidation wasdetermined over 90 minutes. Incubation of EDL and soleus muscles for 90minutes in medium containing 2.5 g/ml gACRP30 leads to a statisticallysignificant increase in oleate oxidation (p<0.05, paired, one-tailed,t-Test) or (p=0.0041, Repeated Measures Analysis of Variance, UnivariateTests of Hypotheses for Within Subject Effects) in both muscle types.

Both muscle types showed a significant response to gACRP30. The relativeincrease in FFA oxidation was 17% (p=0.03) and 10% (p=0.4) for EDL andsoleus, respectively. In humans, muscles represent approximately 25% ofbody weight. Therefore, even a moderate increase in free fatty acidoxidation can have quantitatively important consequences on overallenergy utilization.

Example 13 Effect of gACRP30 on Triglyceride in Muscle and LiverIsolated from Mice

To determine whether the increased FFA oxidation induced by gACRP30 isalso accompanied by increased FFA delivery into muscle or liver, thehindlimb muscle and liver triglyceride content was measured aftergACRP30 treatment of mice. Hind limb muscles as well as liver sampleswere removed from treated and untreated animals and the triglyceride andfree fatty acid concentration was determined following a standard lipidextraction method Shimabukuro, et al., “Direct antidiabetic effect ofleptin through triglyceride depletion of tissues”, Proc Natl Acad SciUSA 94, 4637-4641 (1997) followed by TG and FFA analysis using standardtest kits.

Short-term treatment of animals with gACRP30 (2 injections of 25 μg ofeach given within 3 hours before sacrifice) did not change thetriglyceride content either of hind limb muscle or liver tissue (datanot shown). However, after 3 days of treatment, during which periodnormal C57BL/6J mice consumed a regular rodent diet, mice that hadreceived 25 μg of gACRP30 twice daily showed significantly higher(p=0.002) muscle triglyceride content (FIG. 20A) than those receivingsaline (control: light gray; gACRP30: dark gray). This contrasted with alack of increase in liver triglycerides (FIG. 20B).

Furthermore, no detectable increase in muscle TG was observed after the16-day treatment shown independently by directly measuring the muscle TGcontent and by oil red O staining of frozen microscope sections. Insummary, the data indicate that the increase in TG content wastransient.

These data are consistent with the notion that gACRP30 increases therate of removal of free fatty acids from plasma at least partly byincreasing their delivery to the muscle; much of the FFAs areimmediately oxidized while some are stored as triglycerides andsubsequently oxidized. Further support for this interpretation wasobtained by measuring the concentration of ketone bodies in plasma oftreated and untreated animals following a high fat/sucrose meal.

Ketone bodies (KB) are produced in the liver as a result of free fattyacid oxidation, but KB formation does not occur significantly in muscle.In mice receiving the high fat test meal and saline injection, the levelof plasma KB increased significantly over the next 3 hours (183±12%,n=6). Animals treated with gACRP30, on the other hand, showed noincrease in plasma KB concentrations. Thus, gACRP30 inhibits eitherdirectly KB formation or can decrease KB production by inhibiting liverFFA oxidation.

Example 14 Effect of aACRP30 on Weight Gain and Weight Loss of Mice

Two independent studies showed that gACRP300 also affects overall energyhomeostasis. In the first, 10-week-old male C57BL/6J mice were put on avery high fat/sucrose purified diet for 19 days to promote weight gain(see Example 5); the average body weight at this time was 30 g. The micewere then surgically implanted with an osmotic pump (Alzet. Newark,Del.) delivering either 2.5 μg/day of gACRP30, 5 μg/day of ACRP30, orphysiological saline. The mice were continued on the high fat diet andtheir body weight was recorded over the following 10-day period.

Mice treated with saline or 5 g/day of full-length ACRP30 continued togain weight at an average daily rate of 0.16%, and 0.2%, respectively.In contrast, mice treated with gACRP30 experienced a significant weightreduction (−3.7%, p=0.002) during the first 4 days and then their weightremained constant (FIG. 21A). Thus, in this inbred strain of normalmice, a continuous infusion of a daily low dose of gACRP30 can preventweight gain caused by high fat sucrose feeding, in a sustainable way.

This result was confirmed and extended in a second study performed inmature 9 month old, male obese C57BL/6J mice that had been on the samehigh fat sucrose diet for 6 months; the average body weight when thestudy began was 52.5=0.8 g. Three groups of 8 mice were treated withsaline, ACRP30 or gACRP30 for 16 days. Animals in the treated groupreceived twice daily μg of protein subcutaneously. Body weights wererecorded at the indicated time points.

Treatment with gACRP30 led to significant (p<0.05) weight loss at day 3.This effect became even more significant as the study continued. Duringthe 16 day study period, the obese C57BL/6J mice that received gACRP30lost about 8% (p=0.001) of their initial body weight despite the factthat they were maintained on a high fat/sucrose diet (FIG. 21B). Salinetreated animals showed only marginal fluctuations in their body weight(p=n.s.). Animals treated with the full-length ACRP30, but at a 10-foldhigher dose than that used in the first experiment, also lostsignificant weight (−3.2%, p=0.025). Interestingly, mice treated withgACRP30 continued to lose weight at a steady rate during the 6-day studyperiod, while the rate of weight reduction in those treated with thefull-length ACRP30 decreased during the later phase of the study. Foodconsumption in gACRP30 treated animals was not significantly differentfrom saline or ACRP30 treated animals (FIG. 21D).

Treatment with gACRP30 caused a significant reduction in theconcentration of plasma free fatty acids (FIG. 21C). This effect wassignificant after 3 days of treatment (p<0.05 vs. saline) and continuedthroughout the complete study period. Shown is the plasma FFA level atday 16 of the study. The initial FFA plasma concentration was the samein all three treatment groups. It should be noted, however, that despitethis reduction the plasma free fatty acid concentration of thesemassively obese animals remains about 40-60% higher than that of normalmice. A blood chemistry analysis (including determination of SGPT, SGOT,urea, creatinine or bilirubin) performed on the terminal blood samplesdid not reveal any abnormal plasma parameters (FIG. 22).

Data are expressed throughout as mean±SEM: a p-value<0.05 was consideredstatistically significant. Statistical analysis was typically done usingeither the unpaired Student's r test or the paired Student's t test, asindicated in each study.

Example 15 Detection of APM1 (OBG3) Fragment in Human Plasma AfterImmunoprecipitation

The recombinant form of ACRP30 protein used has an apparent molecularweight of 37 kDa and forms a dimer of 74 kDa (FIG. 23A, Lane II). Aproteolytic fragment that contains the entire globular head region(gACRP30) and that migrates with an apparent molecular weight of 18 kDawas generated using acetylated trypsin (FIG. 23A, lane I). Both proteinpreparations (ACRP30 and gACRP30) were essentially endotoxin free:ActiClean Etox affinity columns (Sterogene Bioseparations Inc. Carlsbad,Calif.) were used to remove potential endotoxin contaminations followingthe manufacturer's protocol. Endotoxin levels were determined byEndosafe, Charleston, S.C. As determined by N-terminal sequencing ofpurified gACRP30, the site of cleavage was just before amino acid 104(just before amino acid 101 for human gOBG3 or APM1).

Immunoprecipitation of human plasma APM1 followed by Western blottingwas used to detect a cleavage product of APM1, the human homolog ofACRP30, using a globular head specific anti-serum for theimmunoprecipitation step as well as for the detection step. Preimmuneserum or serum raised against the globular head domain or humannon-homologous region (HDQETTTQGPGVLLPLPKGA) were cross-linked toprotein A (Sigma Chemical CO, Saint Louis, Mo.) usingdimethyl-pimelimidate-dihydrochlonde (Sigma Chemical Co, Saint Louis,Mo.). After washing (0.2 M salt) proteins were eluted from protein A,separated by SDS-PAGE, transferred to Protran® pure nitrocellulosemembrane (Schleicher and Schuell, Keene. N.H.) using standardprocedures. APM1 products were visualized using globular head domainantibodies labeled with biotin: horseradish peroxidase conjugated toStreptavidin and CN/DAB substrate kit (Pierce, Rockford, Ill.) accordingto manufacturer's instructions.

The apparent molecular weight of this truncated form was 27 kDa,corresponding to about 70% of the complete form of APM1 (FIG. 23B. LaneIV). This truncated form was not detectable when immunoprecipitation wasperformed using a different antibody directed against the humannon-homologous region (HDQETTTQGPGVLLPLPKGA) of APM1; this domain islocated toward the NH₂ terminal end of the protein outside of theglobular domain (FIG. 23, Lane V). Both anti-APM1 antibodies directedagainst either the globular or the non-globular domain identified thefull-length form of the protein, as well as a low abundance dimer ofapparent MW 74 kDa.

Example 16 Effect of gACRP30 on FFA following Intralipid Injection

Two groups of mice (n=5 each) were intravenously (tail vein) injectedwith 30 μL bolus of Intralipid-20% (Clintec) to generate a sudden risein plasma FFAs, thus by-passing intestinal absorption. (Intralipid is anintravenous fat emulsion used in nutritional therapy). A treated group(♦ gACRP30-treated) was injected with gACRP30 (25 μg) at 30 and 60minutes before Intralipid was given, while control animals (▴ control)received saline. Plasma was isolated and FFAs were measured as describedpreviously.

The effect of gACRP30 on the decay in plasma FFAs following the peakinduced by Intralipid injection was then monitored. As shown in FIG. 24,gACRP30 accelerates the removal of FFAs from plasma after Intralipidinjection. Thus, gACRP30 accelerates the clearance of FFAs withoutinterfering with intestinal absorption. Although not wishing to be boundby any theory, because Intralipid does not elicit a significant insulinresponse, the results also indicate that gACRP30 regulation of FFAmetabolism occurs independently of insulin.

Example 17 Solubilization of OBG3 and Fragments Thereof

Vector construction: Polynucleotides encoding for polypeptidescomprising amino acid residues 111-191, 191-244, 101-244, 105-244, or110-244 of APM1 (SEQ ID NO:6) or amino acid residues 104-247 of ACRP30(SEQ ID NO:4) were cloned into bacterial expression vector pTrcHis.Polynucleotides encoding for polypeptides comprising amino acid residues111-191, 191-244, 101-244, 110-244 or 19-244 (full-length) of APM1 (SEQID NO:6) were cloned into a modified bacterial expression vector pET30a(containing His Tag at C-terminal of the AP2M1 polypeptides).Polynucleotides encoding for polypeptides comprising amino acid residues111-191, 191-244, 101-244, 105-244, 110-244, or 19-244 of APM1 (SEQ IDNO:6) or amino acid residues 104-247 of ACRP30 (SEQ ID NO:4) were clonedinto Baculoviral expression vector FastBacHT. Polynucleotides encodingfor polypeptides comprising amino acid residues 111-191, 191-244,101-244, 105-244, or 110-244 of APM1 (SEQ ID NO:6) were cloned intomammalian expression vector pcDNA4HisMax. Polynucleotides encoding forpolypeptides comprising amino acid residues 1-244 or 1-19 and 101-244 ofAPM1 (SEQ ID NO:6) were cloned into mammalian expression vectorpcDNA3.1Hygro. Alternatively, polynucleotides encoding for polypeptidescomprising amino acid residues 1-244, 18-244, 85-244, 101-244, 102-244,103-244, 111-191, 191-244, 144-199, or any fragment of APM1 (SEQ IDNO:6), or amino acid residues 88-247, 104-247, 105-247, 106-247 or anyfragment of ACRP30 (SEQ ID NO:4) are cloned into any bacterial,mammalian, or baculoviral expression vector, preferably selected frompTrcHis, pET30a, FastBacHT, pcDNA4His, or pcDNA3.1Hygro. Alternately,polynucleotides encoding for polypeptides comprising amino acid residues18-244.85-244, 101-244, 102-244, 103-244, 111-191, 191-244, 14″-199, orany fragment of APM1 (SEQ ID NO:6), or amino acid residues 88-247,104-247, 105-247, 106-247 or any fragment of ACRP30 (SEQ ID NO:4) arecloned into Pichia Pastoris (yeast) expression vector, preferablyPHIL-S1.

Cell growth and induction of OBG3 expression: DH5alpha E. coli hostcells were transformed with a pTrcHis expression vector containingpolynucleotides of SEQ ID NO:5 encoding for a polypeptide comprisingamino acid residues 110-214 of APM1 (SEQ ID NO:6), also referred to asgAPM1 (110-244). Cells were grown overnight at room temperature (−22°C.) and induced at an approximate density of 0.45 OD₆₀₀. The cells wereinduced with 1 mM IPTG for 6 hours at room temperature. Cells wereharvested and lysed by French Press and sonication in buffer comprising50 mM Bis-Tris-Propane, pH 8.75, 1% Triton X-100, 5 mM MgCl₂ and 5 mMCaCl₂ and 50 mM NaCl. Extracts were centrifuged to obtain a solublefraction. The soluble protein in the supernatant fraction was passedover a Ni-affinity column at 5° C. The column was then washed with 25 mMimidazole. Alternatively, several washes of increasing imidazole up to25 mM is used. The gAPM1 (110-224) protein was eluted from the columnwith a concentration of 100 mM imidazole. The eluant comprising thegAPM1 (110-244) fraction was run over an ion exchange column using DEAEresin. Alternatively, a hydroxyapatite column is used. The gAPM1(110-244) protein was eluted with high salt buffer and dialyzed toreduce the salt concentration. This material was used in Example 18 toassay affect on FFA oxidation.

Example 18 Effect of gAPM1 on Free Fatty Acid Oxidation

Bacterially expressed APM1-His tag (110-214), gACRP30-his tag (104-247)and gACRP30 (104-247)-N-term-His/C-term-Flag tagged proteins werecompared for activity in a free fatty acid (FFA) oxidation assay.Briefly, C2C12 cells and SKMC cells were grown to ˜95% confluency. Cellswere differentiated for 7 days. Cells were starved overnight in serumfree DME containing 0.5% Albumax I (fatty acid rich BSA). Cells werethen preincubated in medium comprising Basal Medium Eagle (BME), 20 mMHEPES, pH7.4, 4 rmM L-Glutamine, 1% fatty acid free BSA (0.15 mM). (As anegative control, a flask with no cells was included with 2 ml ofPreincubation media alone.) Cells were treated with either 5.0 ug/mlgACRP30 trypsin-cleaved (0.275 mg/ml). 5.0 ug/ml gACRP30 bacteriallyexpressed (0.36 mg/ml), 5.0 ug/ml gACRP30 His-Flag tagged bacteriallyexpressed (0.24 mg/ml), 5.0 ug/ml gAPM1 (110-244) (0.09 mg/ml)bacterially expressed (bacterial expression described in Example 17) for2 hours at 37° C. As a positive control, 100 uM isoproteranol was addedto the cells 15 minutes before addition of perchloric acid.

Treated cells were assayed as follows:

-   -   1. 100 ul of media with 4 mM palmitic acid and 4 uCi/ml        14C-palmitic acid was added to each of the flasks, and cells        were incubated at 37° C., for 90 minutes.    -   2. Using a 1 ml Luer-lok syringe, Solvable was injected into the        center well containing a filter paper approximately 15 min        before adding the perchloric acid.    -   3. Using a 1 ml Luer-lok syringe. 1 ml perchloric acid was added        and cells were incubated for 1 hour at 37° C.    -   4. After 1 hour, 15 ml Hionic Fluor was added and tilter paper        was transferred into appropriate scintillation vial and shaken        for 30 min at 4° C., then counted.

The N-terminally His tagged gAPM1 (110-244), gACRP30 (104-247) andisoproterenol

(positive control) significantly increased FFA oxidation abovenon-treated cells. The recombinant mouse protein containing both anN-term His tag and a C-term FLAG tag was not active in this assay.

Example 19 Assay for Dipentidyl Peptidase Cleavage of OBG3 or cOBG3Polypeptide Fragment

Dipeptidyl peptidase cleavage of gAiPM (85-244) or gAPM1 (103-244)polypeptide fragment is determined by ELISA using a monoclonal antibodythat specifically binds to intact gAPM1 polypeptide fragment but not tosaid gAPM1 polypeptide fragment from which the N-terminal dipeptide EPhas been removed. That is, said antibody binds to gAPM1 (85-241) but notto gAPM1 (binding to S5-244) or said antibody binds to gAPM1 (103-244)but not to gAPM1 (105-244). Dipetidyl peptidase is selected from but notrestricted to human plasma comprised of dipeptidyl peptidase, solublehuman CD26, or soluble human Attractin.

Briefly, gAPM1 (5 μl, 100 fmol) is added to plasma samples (95 μl) andincubated for 1 h at 37° C., after which the amount of gAPM1 polypeptidefragment that remains undegraded is determined by enzyme-linkedimmunosorbent assay (ELISA) using a monoclonal antibody specific forintact gAPM1 polypeptide fragment. As a reference, gAPM1 polypeptidefragment (5 μl, 100 fmol) is added to heat-inactivated plasma (95 μl),which contains 0.01 mmol/l valine-pyrrolidide and 500 KIE/ml aprotinin.By defining the dipeptidyl peptidase activity in the reference as zeroand comparing the amount of intact gAPM1 polypeptide fragment in thesamples with the reference, the extent of dipeptidyl peptidase cleavageof gAPM1 polypeptide fragment is calculated relative to the referenceand expressed as a percentage.

Example 20 In Vivo Evaluation of OBG3 of gOBG3 Polypeptide Fragments ofthe Invention

7-Day Evaluation: A study protocol was established to evaluate theeffects on lipid or glucose metabolism of isolated and purifiedrecombinant OBG3 or gOBG3 polypeptides of the invention, for example,amino acids 101-244, 105-244, 110-244, 19-244, 111-191, 191-244, and144-199 of SEQ ID NO:6, and amino acids 104-247 of SEQ ID NO:4, andHis-tagged analogues thereof. This protocol can be expanded to includethe efficient screening of any polypeptide for potential use in methodsto alter lipid or glucose metabolism. A feature of this protocol is theshort-term experimental length as well as detection of intermediate-termexperimental effects. Mice are treated for a total of 7 days. At thestart of the treatment acute effects either on fat glucose metabolism oron hepatic glucose output are measured by conducting a postprandiallipemia study (PPL) or an Insulin tolerance test (ITT), respectively.Short-term effects on blood chemistry are determined after 3-4 days oftreatment and a complete analysis is performed at the end of thetreatment period. This includes a second study, either PPL. ITT or OGTT(oral glucose tolerance test). In addition effects on body weight, bodycomposition (% lean and fat tissue), fat storage in liver and muscle aremeasured and tissues are isolated to follow effects on gene expressionor to determine histological changes according to routine methods knownin the art.

I. Assay for Glucose Tolerance

A standard Glucose Tolerance Test (GTT) following injection of testpolypeptides is performed on mice as follows:

-   -   1. Mice are fasted for 3-6 hours. The standard model uses        C57Bl/6 male mice kept on a high fat diet, this results in        decreased insulin sensitivity.    -   2. A baseline blood sample is taken from the tail and the        glucose level is measured using glucose test strips and the ‘One        Touch Ultra System’ (Johnson & Johnson), as with all following        blood draws.    -   3. Treatment with test polypeptide (5-150 ug/50 ul/mouse) is        given by i.p, injection. Control animals are injected with equal        volume of saline.    -   4. Immediately following treatment, glucose is injected i.p.        (Dose: 1 g/kg body weight).    -   5. After all injections are done, the plasma glucose level is        monitored at 30′, 60′, 90′, and 120′ post-treatment.    -   6. For the 7-day evaluation, mice are injected at the same time        everyday for 7 consecutive days, and GTT assay is performed on        each day according to step 5 above.

II. Assay for Insulin Tolerance

A standard Insulin Tolerance Test (ITT) following injection of testpolypeptides is performed on mice as follows:

-   -   1. Mice are fasted for 3-6 hours. The standard protocol uses        C57Bl/6 male mice kept on a high fat diet, this results in        decreased insulin sensitivity.    -   2. A baseline blood sample is taken from the tail and the        glucose level is measured using glucose test strips and the ‘One        Touch Ultra System’ (Johnson & Johnson), as with all following        blood draws.    -   3. Treatment with test protein (5-150 ug50 ul/mouse) is given by        i.p, injection. Untreated animals are injected with equal volume        of saline.    -   4. Immediately following treatment insulin is injected (Dose:        0.3 IU/kg body weight).    -   5. The plasma glucose level is monitored at 15′, 30′, 60′, 90′,        and 120′ post-treatment.    -   6. For the 7-day evaluation, mice are injected at the same time        everday for 7 consecutive days, and ITT assay is performed on        each day according to step 5 above.

III. Assay for Postprandial Lipemia

A standard Postprandial lipemia assay (PPL) following injection of testpolypeptides is performed on mice as follows:

-   -   1. Mice are fasted tbr 3-6 hours. The standard protocol uses        normal C57Bl/6 male mice.    -   2. A baseline blood sample is taken from the tail.    -   3. A high fat/high sucrose test meal (6 g butter, 6 g sunflower        oil, 10 g non fat dry milk, 10 g sucrose, 12 ml distilled water        prepared fresh) is given by gavage (Dose: 1% of the animal total        body weight in ml).    -   4. Following the test meal, animals are treated with test        protein (5-150 ug/50 ul/mouse) given by i.p, injection.        Untreated animals are injected with equal volume of saline. This        treatment is repeated each day throughout a 7-day evaluation        study.    -   5. Blood samples are taken at 1, 2, 3 and 4 hrs following        treatment. Samples are immediately stored on ice until plasma        separation.    -   6. Plasma samples are assayed for metabolic parameters including        glucose, insulin, leptin triglycerides, and FFAs.

Plasma glucose concentration is determined as described above in the GTTassay using the ‘One Touch Ultra System’. Alternatively, a standardglucose test kit is used (Sigma). The concentration of triglycerides andfree fatty acids (FFAs) are determined using calorimetric assays(Triglycerides. Sigma: FFA, Wako Biochemicals, Osaka). Insulin andLeptin are determined by RLA (Linco Research. St Charles, Mo.).

In addition to the 7-day evaluation study protocol, GTT, ITT, and PPLassays can be performed in any length study, including single day,short-term, and long-term periods following injection of the testpolypeptides.

Example 21 Infant Formula Supplementation

The following example describes a method of administering OBG3 or gOBG3polypeptides to newborns as supplemental nutritional support and furtherprovides a method of promoting growth of an infant by administering OBG3or gOBG3-fortified human breast milk, or OBG3 or gOBG3-fortified breastmilk substitute formulation from a nonhuman source. Dehydrated orlyophilized OBG3 or gOBG3 polypeptide powder is directly added to pumpedhuman breast milk (freshly pumped or prewarmed after storage) or anyprewarmed breast milk substitute formulation from a nonhuman source, ina range of 5-1000 ng/ml, preferably 20-800 ng.ml, more preferably 65-650ng/ml. Supplementation with polypeptides of the invention is provided toinfants, particularly preterm infants, in bottle feedings of humanbreast milk or breast milk substitute at even feeding throughout theday, and is continued to be provided from birth to 6 months of ace.Preterm infants may be of low birth weight or very low birth weight.

A primary objective of the study is to demonstrate that a polypeptide ofthe invention added to human milk (HM) or human milk substitute (HMS)supports acceptable growth in preterm infants. A second objective is toevaluate the serum biochemistries (i.e., protein status, calcium,alkaline phosphatase), tolerance, clinical problems, and morbidity ofpremature infants consuming the nutritional module. Another secondaryobjective is to compare the supplemental composition of the instantinvention to a commercial fortifier powder that has been in use for anumber of years to promote growth in preterm infants. Anintent-to-treat, prospective, randomized, double-blinded multicenterstudy is conducted to evaluate preterm infants receiving preterm milksupplemented with either a commercially available powdered human milkfortifier (Enfamil® Human Milk Fortifier, control) or the supplementalpolypeptide (test) powder of the current invention (experimental) atevery feeding. Subjects are enrolled and randomized to each fortifierpowder prior to 21 days of life. Study Day 1 is when fortification ofthe test powder begins and the subject reaches an intake of at least 100mL/kg/day. Anthropometric indices, serum biochemistries, intake,tolerance, and morbidity data are assessed. Each infant is studied untilhospital discharge; only anthropometric variables (weight, length, andhead circumference) are collected after Study Day 29. Premature infantsare recruited from neonatal intensive care units that had agreed tocollaborate with study investigators. Single, twin, or triplet infantsborn around 33 weeks gestational age, with appropriate weight forgestational age, and weighing around 1600 g are eligible to participate.One hundred and forty-four infants are randomized to either control orexperimental; 70 preterm infants are randomized to the control group and74 preterm infants are randomized to the experimental group. Therandomization is proportional for birth weight and gender.

The independent variables (treatments) are the control fortifier powderand the experimental test powder which are added to HM or HMS. Bothfortifiers (test and control) are provided in small packets in powderedform and are added to 25 mL HM or HMS. The primary outcome variable isweight gain (g/kg/day) from study day 1 to study day 29 or discharge,whichever comes first. Secondary outcome variables are length gain(mm/day) and serum biochemistries to evaluate protein status,electrolyte status, mineral homeostasis, and vitamin A and E status.Serum biochemistries also include unscheduled laboratory results to berecorded in the medical chart. Tertiary variables include headcircumference gain (mm/day), clinical history, intake, tolerance,clinical problems/morbidity, respiratory status, antibiotic use, and thenumber of transfusions. Mean total energy intakes during the studyperiod is not different between the groups, around 118 kcal/kg/day.

INCORPORATION OF RELATED APPLICATIONS

This application hereby incorporates each of the provisionalapplications, non-provisional applications, and applications filed underthe Patent Cooperation Treaty, as listed on the Application Data Sheetthat is associated with the subject application, by reference and intheir entireties, including any figures, tables, nucleic acid sequences,amino acid sequences, and/or drawings.

ADDITIONAL REFERENCES

-   1. Shapiro, L. & Scherer, P. E., “The crystal structure of a    complement-1q family protein suggests an evolutionary link to tumor    necrosis factor”, Curr Biol 8, 335-338 (1998).-   2. Kishore, U. & Reid, K. B., “Modular organization of proteins    containing Clq-like globular domain”, Immunopharmacology 42, 15-21    (1999).-   3. Kavety, B. & Morgan, J. I., “Characterization of transcript    processing of the gene encoding precerebellin-1”, Brain Res Mol    Brain Res 63, 98-104 (1998).-   4. Satoh. F, et al. “Cerebellin and cerebellin mRNA in the human    brain, adrenal glands and the tumour tissues of adrenal tumour,    ganglioneuroblastoma and neuroblastoma”, J Endocrinol 154, 27-34    (1997).-   5. Mazzocchi, G, et al., “Cerebellin enhances in vitro secretory    activity of human adrenal gland”. J Clin Endocrinol Metab 84,    632-635 (1999).-   6. Kondo. N. & Kondo, J. “Identification of novel blood proteins    specific for mammalian hibernation”. J Biol Chem 267, 473-478    (1992).-   7. Takamatsu. N. Ohba. K. Kondo, J., Kondo, N. & Shiba, T.,    “Hibernation-associated gene regulation of plasma proteins with a    collagen-like domain in mammalian hibernators”, Mol Cell Biol 13,    1516-1521 (1993).-   8. Spiegelman. B. 11. & Hotamtsligil. G. S., “Through thick and    thin: wasting, obesity, and TNF alpha”. Cell 73, 625-627 (1993).-   9. Saito. K, et al. “Regulation of gelatin-binding protein 28    (GBP2S) gene expression by C. EBP”. Biol Pharm Bull 22, 1158-1162    (1999).-   10. Takamatsu. N, et al. “Expression of multiple alpha    1-antitrypsin-like genes in hibernating species of the squirrel    family”. Gene 204, 127-132 (1997).-   11. Ross, S. R., Graves, R. A. & Spiegelman, B. M.“Targeted    expression of a toxin gene to adipose tissue: transgenic mice    resistant to obesity”, Genes Dev 7, 1318-1324 (1993).-   12. Moitra. J, et al., “Life without white fat: a transgenic mouse”,    Genes Dev 12, 3168-3181 (1998).-   13. Peters. S. J. Dyck. D. J. Bonen, A. & Spriet. L. L. “Effects of    epinephrine on lipid metabolism in resting skeletal muscle”, Am J    Physiol 275, E300-309 (1998).-   14. Rigotti, A., Acton. S. L. & Krieger, M., “The class B scavenger    receptors SR-B1 and CD36 are receptors for anionic phospholipids”, J    Biol Chem 270, 16221-16224 (1995).-   15. Hirsch, D., Stahl, A. & Lodish. H. F., “A family of fatty acid    transporters conserved from mycobacterium to man”, Proc Natl Acad    Sci USA 95, 8625-8629 (1998).-   16. Hotamisligil, G. S, et al., “IRS-1-mediated inhibition of    insulin receptor tyrosine kinase activity in TNT-alpha- and    obesity-induced insulin resistance”. Science 271, 665-668 (1996).-   17. Peraldi. P. & Spiegelman, B., “TNT-alpha and insulin resistance:    summary and future prospects”, Mol Cell Biochem 182, 169-175 (1998).-   18. Kirchgessner. T. G., Uysal, K. T., Wiesbrock, S. M.    Marino, M. W. & Hotamisligil, G. S. “Tumor necrosis factor-alpha    contributes to obesity-related hyperleptinemia by regulating leptin    release from adipocytes”, J Clin invest 100, 2777-2782 (1997).-   19. Uysal. K. T., Wiesbrock. S. M., Marino, M. W. & Hotamisligil, G.    S., “Protection from obesity-induced insulin resistance in mice    lacking TNF-alpha function”, Nature 389, 610-614 (1997).-   20. Jensen, D. R. et al. “Prevention of diet-induced obesity in    transgenic mice overexpressing skeletal muscle lipoprotein lipase”.    Am J Physiol 273, R683-689 (1997).-   21. Levak-Frank, S, et al. “Muscle-specific overexpression of    lipoprotein lipase causes a severe myopathy characterized by    proliferation of mitochondria and peroxisomes in transgenic mice”. J    Clin Invest 96, 976-986 (1995).-   22. Scheja, L, et al. “Altered insulin secretion associated with    reduced lipolytic efficiency in aP2−/−mice”, Diabetes 48, 1987-1994    (1999).-   23. Griffin. M. E, et al. “Free fatty acid-induced insulin    resistance is associated with activation of protein kinase C theta    and alterations in the insulin signaling cascade”. Diabetes 48.    1270-1274 (1999).-   24. Dresner, A, et al. “Effects of free fatty acids on glucose    transport and IRS-1-associated phosphatidylinositol 3-kinase    activity”. J Clin Invest 103, 253-259 (1999).-   25. Kelley. D. E. Goodpaster. B. Wing. R. R. & Simoneau. J. A.    “Skeletal muscle fatty acid metabolism in association with insulin    resistance, obesity, and weight loss”. Am J Physiol 277, E1130-1141    (1999).-   26. Roden, M, et al. “Mechanism of free fatty acid-induced insulin    resistance in humans”, J Clin Invest 97, 2859-2865 (1996).-   27. Deacon. C. F, et al. “Dipeptidyl peptidase IV inhibition reduces    the degradation and clearance of GIP and potentiates its    insulinotropic and antihyperglycemic effects in anesthetized pigs”.    Diabetes 50, 1588-1597 (2001).

1. An isolated polypeptide consisting of consecutive amino acids: 87-244of SEQ ID NO: 6, 88-244 of SEQ ID NO: 6, 89-244 of SEQ ID NO: 6, 90-244of SEQ ID NO: 6, 91-244 of SEQ ID NO: 6, 92-244 of SEQ ID NO: 6, 93-244of SEQ ID NO: 6, 94-244 of SEQ ID NO: 6, 95-244 of SEQ ID NO: 6, 96-244of SEQ ID NO: 6, 97-244 of SEQ ID NO: 6, 98-244 of SEQ ID NO: 6, 99-244of SEQ ID NO: 6, 100-244 of SEQ ID NO: 6, 111-191 of SEQ ID NO: 6,144-199 of SEQ ID NO: 6, 191-244 of SEQ ID NO: 6, 108 to 244 of SEQ IDNO: 6, amino acids 110 to 244 of SEQ ID NO: 6, amino acids 108 to 244 ofSEQ ID NO: 6 to which an N-terminal methionine has been added, or aminoacids 110 to 244 of SEQ ID NO: 6 to which an N-terminal methionine hasbeen added.
 2. The isolated polypeptide according to claim 1, whereinsaid polypeptide consists of consecutive amino acids 87-244 of SEQ IDNO:
 6. 3. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 88-244 of SEQ ID NO: 6.4. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 89-244 of SEQ ID NO: 6.5. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 90-244 of SEQ ID NO: 6.6. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 91-244 of SEQ ID NO: 6.7. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 92-244 of SEQ ID NO: 6.8. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 93-244 of SEQ ID NO: 6.9. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 94-244 of SEQ ID NO: 6.10. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 95-244 of SEQ ID NO: 6.11. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 96-244 of SEQ ID NO: 6.12. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 97-244 of SEQ ID NO: 6.13. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 98-244 of SEQ ID NO: 6.14. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 99-244 of SEQ ID NO: 6.15. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 100-244 of SEQ ID NO: 6.16. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 111-191 of SEQ ID NO: 6.17. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 144-199 of SEQ ID NO: 6.18. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 191-244 of SEQ ID NO: 6.19. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 108-244 of SEQ ID NO: 6.20. The isolated polypeptide according to claim 1, wherein saidpolypeptide consists of consecutive amino acids 108 to 244 of SEQ ID NO:6 to which an N-terminal methionine has been added.
 21. The isolatedpolypeptide according to claim 1, wherein said polypeptide consists ofconsecutive amino acids 110 to 244 of SEQ ID NO: 6 to which anN-terminal methionine has been added.
 22. The isolated polypeptideaccording to claim 1, wherein said polypeptide consists of consecutiveamino acids 110 to 244 of SEQ ID NO:
 6. 23. A composition comprising acarrier and a polypeptide consisting of consecutive amino acids: 87-244of SEQ ID NO: 6, 88-244 of SEQ ID NO: 6, 89-244 of SEQ ID NO: 6, 90-244of SEQ ID NO: 6, 91-244 of SEQ ID NO: 6, 92-244 of SEQ ID NO: 6, 93-244of SEQ ID NO: 6, 94-244 of SEQ ID NO: 6, 95-244 of SEQ ID NO: 6, 96-244of SEQ ID NO: 6, 97-244 of SEQ ID NO: 6, 98-244 of SEQ ID NO: 6, 99-244of SEQ ID NO: 6, 100-244 of SEQ ID NO: 6, 111-191 of SEQ ID NO: 6,144-199 of SEQ ID NO: 6, 191-244 of SEQ ID NO: 6, 108 to 244 of SEQ IDNO: 6, amino acids 110 to 244 of SEQ ID NO: 6, amino acids 108 to 244 ofSEQ ID NO: 6 to which an N-terminal methionine has been added, or aminoacids 110 to 244 of SEQ ID NO: 6 to which an N-terminal methionine hasbeen added.
 24. The composition according to claim 23, wherein saidpolypeptide consists of consecutive amino acids 87-244 of SEQ ID NO: 6.25. The composition according to claim 23, wherein said polypeptideconsists of consecutive amino acids 88-244 of SEQ ID NO:
 6. 26. Thecomposition according to claim 23, wherein said polypeptide consists ofconsecutive amino acids 89-244 of SEQ ID NO:
 6. 27. The compositionaccording to claim 23, wherein said polypeptide consists of consecutiveamino acids 90-244 of SEQ ID NO:
 6. 28. The composition according toclaim 23, wherein said polypeptide consists of consecutive amino acids91-244 of SEQ ID NO:
 6. 29. The composition according to claim 23,wherein said polypeptide consists of consecutive amino acids 92-244 ofSEQ ID NO:
 6. 30. The composition according to claim 23, wherein saidpolypeptide consists of consecutive amino acids 93-244 of SEQ ID NO: 6.31. The composition according to claim 23, wherein said polypeptideconsists of consecutive amino acids 94-244 of SEQ ID NO:
 6. 32. Thecomposition according to claim 23, wherein said polypeptide consists ofconsecutive amino acids 95-244 of SEQ ID NO:
 6. 33. The compositionaccording to claim 23, wherein said polypeptide consists of consecutiveamino acids 96-244 of SEQ ID NO:
 6. 34. The composition according toclaim 23, wherein said polypeptide consists of consecutive amino acids97-244 of SEQ ID NO:
 6. 35. The composition according to claim 23,wherein said polypeptide consists of consecutive amino acids 98-244 ofSEQ ID NO:
 6. 36. The composition according to claim 23, wherein saidpolypeptide consists of consecutive amino acids 99-244 of SEQ ID NO: 6.37. The composition according to claim 23, wherein said polypeptideconsists of consecutive amino acids 100-244 of SEQ ID NO:
 6. 38. Thecomposition according to claim 23, wherein said polypeptide consists ofconsecutive amino acids 111-191 of SEQ ID NO:
 6. 39. The compositionaccording to claim 23, wherein said polypeptide consists of consecutiveamino acids 144-199 of SEQ ID NO:
 6. 40. The composition according toclaim 23, wherein said polypeptide consists of consecutive amino acids191-244 of SEQ ID NO:
 6. 41. The composition according to claim 23,wherein said polypeptide consists of consecutive amino acids 108-244 ofSEQ ID NO:
 6. 42. The composition according to claim 23, wherein saidpolypeptide consists of consecutive amino acids 108 to 244 of SEQ ID NO:6 to which an N-terminal methionine has been added.
 43. The compositionaccording to claim 23, wherein said polypeptide consists of consecutiveamino acids 110 to 244 of SEQ ID NO: 6 to which an N-terminal methioninehas been added.
 44. The composition according to claim 23, wherein saidpolypeptide consists of consecutive amino acids 110 to 244 of SEQ ID NO:6.
 45. A method of reducing free fatty acid levels in an individualcomprising administering an effective amount of a composition comprisingcarrier and a polypeptide consisting of consecutive amino acids: 87-244of SEQ ID NO: 6, 88-244 of SEQ ID NO: 6, 89-244 of SEQ ID NO: 6, 90-244of SEQ ID NO: 6, 91-244 of SEQ ID NO: 6, 92-244 of SEQ ID NO: 6, 93-244of SEQ ID NO: 6, 94-244 of SEQ ID NO: 6, 95-244 of SEQ ID NO: 6, 96-244of SEQ ID NO: 6, 97-244 of SEQ ID NO: 6, 98-244 of SEQ ID NO: 6, 99-244of SEQ ID NO: 6, 100-244 of SEQ ID NO: 6, 111-191 of SEQ ID NO: 6,144-199 of SEQ ID NO: 6, 191-244 of SEQ ID NO: 6, 108 to 244 of SEQ IDNO: 6, amino acids 110 to 244 of SEQ ID NO: 6, amino acids 108 to 244 ofSEQ ID NO: 6 to which an N-terminal methionine has been added, or aminoacids 110 to 244 of SEQ ID NO: 6 to which an N-terminal methionine hasbeen added.